E-cadherin-based complexes regulate cell behaviour

ARTICLES
Distinct E-cadherin-based complexes regulate cell
behaviour through miRNA processing or Src and
p120 catenin activity
Antonis Kourtidis1
, Siu P. Ngok1,5
, Pamela Pulimeno2,5
, Ryan W. Feathers1
, Lomeli R. Carpio1
, Tiffany R. Baker1
,
Jennifer M. Carr1
, Irene K. Yan1
, Sahra Borges1
, Edith A. Perez3
, Peter Storz1
, John A. Copland1
, Tushar Patel1
,
E. Aubrey Thompson1
, Sandra Citi4
and Panos Z. Anastasiadis1,6
E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here,
we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed
polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and
a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas
of cell–cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is
PLEKHA7 dependent. The PLEKHA7–microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and
possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to
suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our
work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising
association with the microprocessor.
p120 catenin (p120) was identified as a tyrosine phosphorylation
substrate of the Src oncogene1
and an essential component of the
cadherin complex2
. The interaction with p120 stabilizes E-cadherin
junctional complexes by preventing E-cadherin endocytosis2–5
.
p120 also regulates the activity of Rho-GTPases, and thus the
organization of the actomyosin cytoskeleton6–9
. By stabilizing
E-cadherin, p120 is expected to act as a tumour suppressor, and
mouse knockout studies support this notion10
. However, p120 also
exhibits tumour-promoting activities, as an essential mediator of
anchorage-independent growth and cell migration induced by EGFR,
HER2, Rac1 or Src (refs 11–13). This was partly attributed to the
expression of different cadherin family members14,15
; however, p120
can induce tumour growth even in the presence of E-cadherin13,16
and is the essential intermediate for E-cadherin-mediated Rac1
activation and subsequent proliferation induction17
. Consistent
with this, E-cadherin is still expressed in several types of aggressive
and metastatic cancer18–20
. Therefore, despite their significance
in epithelial adhesion and cellular regulation, present knowledge
on the role of E-cadherin and p120 in cancer is conflicting
and inconclusive.
In the present study, we sought to reconcile the apparently
contradictory observations and clarify the roles of p120 and
E-cadherin in epithelial cell behaviour. Recently, the p120 binding
partner PLEKHA7 was shown to specifically localize at the apical
zonula adherens (ZA) but not along lateral surfaces of epithelial cells,
as for p120 or E-cadherin21,22
. By using PLEKHA7 as a marker of the
apical ZA in mature epithelial cells, we characterize two distinct p120-
associated complexes with antagonistic functions and we describe a
microRNA (miRNA)-mediated mechanism through which the ZA
suppresses transformed cell growth.
1
Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, 4500 San Pablo Road, Jacksonville, Florida 32224, USA. 2
Department of
Molecular Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211, Geneva 4, Switzerland. 3
Division of Hematology/Oncology, Mayo Clinic, 4500 San Pablo
Road, Jacksonville, Florida 32224, USA. 4
Department of Cell Biology and Institute of Genetics and Genomics of Geneva, University of Geneva, 30 quai
Ernest-Ansermet, CH-1211, Geneva 4, Switzerland. 5
Present addresses: Division of Hematology, Oncology, Stem Cell Transplantation and Cancer Biology, Department
of Pediatrics, Stanford School of Medicine, Stanford, California 94305-5457, USA (S.P.N.); Department of Genetics and Complex Diseases, Harvard T. Chan School of
Public Health, 665, Huntington Avenue, Boston, Massachusetts 02115, USA (P.P.).
6
Correspondence should be addressed to P.Z.A. (e-mail: panos@mayo.edu)
Received 19 March 2015; accepted 20 July 2015; published online 24 August 2015; DOI: 10.1038/ncb3227
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1
© 2015 Macmillan Publishers Limited. All rights reserved

ARTICLES
RESULTS
Two distinct p120-associated populations exist at epithelial
junctions
Double immunofluorescence (IF) carried out in intestinal (Caco2)
and renal (MDCK) polarized monolayers confirmed previous results
that PLEKHA7 co-localizes with p120 or E-cadherin only in a narrow
area apically at the junctions, whereas p120 and E-cadherin are also
found basolaterally (Fig. 1a and Supplementary Fig. 1a–c; refs 21,
22). The ZA markers afadin, circumferential actin and myosin IIA
(refs 23,24) co-localized precisely with PLEKHA7 (Supplementary
Fig. 1d), as previously shown22
, verifying that PLEKHA7 labels the ZA
in these monolayers.
Unlike PLEKHA7, tyrosine phosphorylation of p120 at the Src-
targeted sites Tyr 96 and Tyr 228 (ref. 25), which has been associated
with cancer11,26,27
, was abundant basolaterally but not apically
(Fig. 1b and Supplementary Fig. 1e,f). In contrast, phosphorylation
of p120 at the non-Src-targeted Thr 310 site was both apical and
basolateral (Supplementary Fig. 1g). Total Src was distributed both
basolaterally and apically (Fig. 1c), although active Src, denoted
by auto-phosphorylation at Tyr 416, was absent from the ZA but
present at basolateral areas of cell–cell contact (Fig. 1d), mirroring
the distribution of tyrosine-phosphorylated p120. Furthermore,
p130CAS, a Src target associated with increased cell mobility and
decreased junction stability28
, was excluded from the ZA and was
abundant basolaterally (Fig. 1e).
We also examined the localization of total and active Rho and
Rac by co-staining with PLEKHA7. Total RhoA and Rho-GTP
were restricted at the ZA, whereas total Rac1 and Rac-GTP were
excluded from the ZA and were detected along basolateral cell–
cell contacts (Fig. 1f,g and Supplementary Fig. 1h–j). p190RhoGAP,
a Src substrate that suppresses RhoA activity29
, was also excluded
from the ZA (Fig. 1h), further indicating that Rac1 and RhoA
activities are mutually exclusive along areas of cell–cell contact.
Together, the data suggest the presence of an apical p120 complex
at the ZA associated with PLEKHA7, and a basolateral complex
lacking PLEKHA7 and encompassing markers associated with a more
aggressive cellular phenotype.
Proteomics identify two distinct junctional complexes in
polarized epithelial cells
To obtain further evidence for the existence of two distinct
junctional complexes, we biochemically separated them on the basis
of the strictly ZA-specific PLEKHA7 localization and the p120
localization throughout cell–cell contacts in polarized cells (Fig. 1a and
Supplementary Fig. 1a–c; ref. 22). Fully polarized Caco2 monolayers
were subjected to intracellular chemical cross linking to maintain the
interactions and then to either direct p120 immunoprecipitation (IP)
to isolate total p120, or a two-step IP, initially with PLEKHA7 to isolate
the apical complex, followed by a sequential p120 IP to isolate the
remaining p120 complexes (Fig. 2a). Western blot and silver staining of
these IPs indicated an efficient separation of the immunoprecipitated
proteins into two fractions (Fig. 2b). Mass spectrometry analysis
revealed 114 putative interactions, of which 47 were uniquely
associated with the apical fraction, 35 with the basolateral and 32 were
common (Fig. 2c and Supplementary Table 1). The apical complex
was enriched in structural and cytoskeletal proteins, such as actin
(ACTB), CKAP5, IQGAP1, ANXA2, MYL6, FLNA and ACTN1. In
contrast, the basolateral complex contained proteins involved in cell
cycle progression (CCAR1, CDC7, CDK5RAP2), signalling (S100A7,
S100A8), endocytosis (VPS33B, VIPAR) and cancer (MCC; Fig. 2c and
Supplementary Table 1).
Western blot of the isolated lysates confirmed the efficient
depletion of PLEKHA7 from the basolateral complex and the
successful IP of p120 by PLEKHA7 in the apical fraction (Fig. 2d).
pY228-p120 was absent from the apical complex lysates but abundant
in the basolateral ones (Fig. 2d), in agreement with the IF data
(Fig. 1b and Supplementary Fig. 1e). Western blot of the separated
fractions also confirmed that E-cadherin, together with α- and
β-catenin, co-precipitates with the apical complex (Supplementary
Fig. 2a), as suggested by our IF data (Supplementary Fig. 1b,c)
and previously published results21
. Indeed, PLEKHA7 localization to
the junctions is E-cadherin and p120 dependent, as indicated by
loss of junctional PLEKHA7 after E-cadherin or p120 knockdown
(Supplementary Fig. 2b,c). Furthermore, only full-length murine
mp120-1A (1A), but not the mp120-4A (4A) isoform that lacks
the amino-terminal PLEKHA7-binding domain2,21
, was able to
rescue junctional localization of PLEKHA7 in p120-depleted cells
(Supplementary Fig. 2c).
In full agreement with the proteomics data, TRIM21, IQGAP1,
ACTN1 and HNRNPA2B1 were detected only in the apical or total
p120 lysates, EWS in all lysates and CCAR1 solely in the basolateral
(Fig. 2d). In addition, IF of polarized cells showed strict apical
junctional co-localization of IQGAP1 with PLEKHA7 (Fig. 2e) and
co-localization of G3BP1 with p120 along both apical and basolateral
junctions (Fig. 2f), further validating the proteomics results (Fig. 2c).
Together, the data confirm the existence of two distinct junctional
complexes in polarized epithelial cells.
PLEKHA7 suppresses anchorage-independent growth and
expression of transformation-related markers
To define the functional role of the apical complex, we carried
out short hairpin RNA (shRNA)-mediated knockdown of PLEKHA7
(Supplementary Fig. 3a). Impedance measurements during junction
assembly of PLEKHA7-depleted cells showed both a delay in resistance
development and an overall lower transepithelial resistance of the
mature monolayer (Fig. 3a). PLEKHA7 knockdown also resulted
in fragmented circumferential actin (Fig. 3b) and a more flattened
cellular phenotype (Supplementary Fig. 3b). These results show that
depletion of PLEKHA7 compromises the integrity of the apical ZA, as
reported earlier21
.
We then examined the effects of disrupting the ZA on the
ability of Caco2 cells to form colonies on soft agar, an established
assay for anchorage-independent growth. PLEKHA7-depleted
cells showed a threefold increase in colony formation (Fig. 3c
and Supplementary Fig. 3c). Furthermore, PLEKHA7 knockdown
increased p120 phosphorylation at Tyr 96 and Tyr 228, Src
phosphorylation at Tyr 416, and p130CAS phosphorylation at
Tyr 165 (Fig. 3d,e and Supplementary Fig. 3d). To account for the
morphological changes and induction of growth on PLEKHA7
depletion, we also examined the levels of transformation-related
and mesenchymal markers15,30,31
. PLEKHA7 knockdown resulted
in increased expression of SNAI1, cadherin 11 (Cad11), cyclin
2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

ARTICLES
p190PLEKHA7 Merged
x
Mergedp120PLEKHA7
y
x
z
x
x
z
p120: Tyr228PLEKHA7
SrcPLEKHA7 Src: Tyr 416PLEKHA7
ApicalBasal
x
x
y
x
y
p130CASPLEKHA7
ApicalBasal
x
y
x
z
Apical
a b
c d
e f
g h
Basal
MergedPLEKHA7 Rho-GTP
y
Merged
Merged
Merged
x
y
Merged
z
x
x
z
y
x
MergedPLEKHA7
ApicalBasal
Rac-GTP
x
y
z
ApicalBasalApicalBasalApicalBasalBasalApical
Figure 1 Polarized epithelial cells show distinct p120-associated populations
at the junctions. Caco2 cells were grown for 21 days to polarize and
subjected to IF for PLEKHA7 and (a) p120, (b) phosphorylated p120
Tyr 228, (c) Src, (d) phosphorylated Src Tyr 416; (e) p130CAS and
(h) p190RhoGAP. Also, Caco2 cells were transfected with (f) a green
fluorescent protein (GFP)–rGBD (rhotekin RhoA-binding domain) construct
to detect active Rho (Rho-GTP) or (g) a yellow fluorescent protein (YFP)–
PBD (PAK-binding domain) construct to detect active Rac (Rac-GTP), and
co-stained with PLEKHA7. In all cases, stained cells were imaged by
confocal microscopy and image stacks were acquired, covering the entire
polarized monolayer between the basal and the apical level. Representative
x–y image stacks and merged composite x–z images are shown. Enlarged
parts of merged images in f and g indicate areas of cell–cell contact. Scale
bars for x–y images, 20 µm; for x–z images, 5 µm; for enlarged parts of f
and g, 3 µm. PLEKHA7 background staining in f and g is an artefact of
paraformaldehyde fixation.
D1 (CCND1), MYC, FAK and VIM (Fig. 3f and Supplementary
Fig. 3d), whereas re-expression of PLEKHA7 reversed these effects
and suppressed p120 phosphorylation (Fig. 3g). Indeed, ectopic
overexpression of SNAI1 in Caco2 cells at modest levels was adequate
to induce a twofold increase in soft agar colony formation (Fig. 3h
and Supplementary Fig. 3e).
Knockdown of NEZHA, the intermediate link between PLEKHA7
and the microtubules21
, retained PLEKHA7 at the ZA but failed to
phenocopy the effects of PLEKHA7 depletion on marker expression
(Supplementary Fig. 3f,g), suggesting that microtubules are not
involved in the observed effects. Next, we tested whether the effects of
PLEKHA7 knockdown are due to its loss from the ZA, by removing
PLEKHA7 from the junctions but retaining its total levels in the
cells. To do this, we transfected Caco2 cells with a p120 mutant
that cannot bind E-cadherin and is cytoplasmic, because it lacks
the first armadillo repeat (mp120- ARM1; ref. 2), but can bind
PLEKHA7, because it contains the N-terminal PLEKHA7-binding
domain21
. Expression of this construct sequestered PLEKHA7 to the

ARTICLES
116
79
52
37
29
17
Then
Grow Caco2s
for 21 days
to polarize
Crosslink
complexes
Lyse,
IP with:
p120 (total p120 to control)
PLEKHA7:
apical
complex
p120:
basolateral
complex
IPs
IgGPLEKH
A7Totalp120
Basolateralp120
199
Mr (K)
Silver staina
c
d
e
f
b
WBs
TRIM21
CCAR1
PLEKHA7
p120
IPs
EWS
HNRNPA2B1
MergedG3BP1p120
x
y
Caco2
ApicalBasal
MergedIQGAP1PLEKHA7
x
y
ACTN1
p-p120: pY228
IQGAP1
Input
IgGPLEKH
A7
Totalp120
Basolateralp120
ApicalBasal
CFTR
FLG2
CLTC
SSR1
PLUNC
PPL
ACTB
ARPC5
ANXA5
ACTN1
DYNC1H1
CKAP5
MYL6
FLNA
TRIM21
EIF3DHSPA8
HSPA9
DCP1B
NOS1
DDX1
DDX4
EIF3A
RALY
FEM1A
ATP5C1 ALDH18A1 GAPDH
WDR82
THOC4NONODDX17
EWS YWHAE
KL
FUS
SFPQ
G3BP2
SLC25A1
MLEC
MSH5
C14orf166SELT
CDK5RAP2
APOA4
AHCYSCYL2
G3BP1
ETAA1
TRIM10
FZD2
LRP1
ODZ2
ANXA2
ANXA4
MYO1D MCCAHNAK
ANXA1 SNRPB
OSTM1
VAV2
CTNNB1
DSG1
FAT2
TTN
JUP
DSP
VIPAR
GLG1
CDC7
WDR67
KPRP
SLC25A5
RELN
DNAH7
SYNE1
LKAP
SERPINB12
VPS35
SHROOM3
ODZ3
CCAR1
CDH1
S100A8
S100A7
BasolateralApical
VPS33B
CTNNA1SPASTIN
PSPC1
PGLS
SLC25A3
CAPRIN1
DLGAP1
NCOA3
CYB5R3
NDUFV1
PITPNB
CES1
DPP4
TMEM128
IQGAP1
HPCAL1
HSP90A1
HNRNPA2B1
HNRNPH3
HNRNPA3
HNRNPA1L2
HSPA1A
PPP1CA
CLYBL
Figure 2 Biochemical separation of two distinct junctional complexes
by proteomics. (a) Outline of the methodology to isolate the apical and
basolateral complexes from polarized Caco2 cells. (b) SDS–polyacrylamide
gel electrophoresis (SDS–PAGE) and silver stain of the isolated junctional
fractions; green arrowheads indicate examples of apical-specific bands,
whereas red arrowheads indicate basolateral-specific bands. (c) Schematic
representation of the proteins identified after mass spectrometry of the
isolated junctional fractions, identified in the apical (green outlines), the
basolateral (red) or both fractions (yellow). Grey connections indicate known
protein–protein interactions. See Supplementary Table 1 for the list of
proteins and mass spectrometry peptide counts. (d) Western blot (WB) of
the lysates from the separated fractions for PLEKHA7, p120 and markers
identified in the apical (TRIM21, IQGAP1, ACTN1, HNRNPA2B1), the
basolateral (CCAR1) and both fractions (EWS). The isolated fractions were
also blotted for phosphorylated p120 Tyr 228 (p-p120: pY228), another
basolateral marker (Fig. 1b). See Supplementary Fig. 8 for unprocessed blot
scans. (e,f) Polarized Caco2 cells were stained and imaged as in Fig. 1 for
PLEKHA7 and IQGAP1 (e) and for p120 and G3BP1 (f). All scale bars: 20 µm.
cytoplasm without altering its total levels and phenocopied the effects
of PLEKHA7 depletion on the levels of the same markers examined
above (Supplementary Fig. 3h,i). Therefore, it is the disruption of
the PLEKHA7-associated apical ZA complex that results in induction
of growth-related signalling. Consistent with this, examination of
the expression and localization of PLEKHA7, p120 and E-cadherin

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Junctionalresistance
(normalizedunits)
h
Junctional integrity
PLEKHA7 shRNA
NT
0
1
2
3
4
Foldchange
Foldchange
PLEKHA7 Actin
PLEKHA7 shRNA
NT
No. 8
PLEKHA7
p120
p-p120: Y228
p-p120: Y96
Actin
Src
p-Src: Y416
NT
PLEKHA7 shRNA
No. 10
Actin
MYC
Cad11
SNAI1
CCND1
VIM
FAK
p130CAS
p-p130CAS: Y165
Actin
PLEKHA7 shRNA
No. 8 No. 10NT
PLEKHA7 shRNA
No. 8NT
PLEKHA7 shRNA
No. 10
∗
∗
SNAI1
PLEKHA7
CCND1
MYC
Actin
p120
p-p120: pY228
LZRS-PLEKHA7
Cad11
p-p120: pY96
0
20
40
60
80
100
120
a
c
f g
h
d e
b
Percentagechange
Apical actin
ring intensity
∗
∗
∗
∗ ∗ ∗
∗
PLEKHA7
PLEKHA7
PLEKH
A7
shRN
A
N
T
0
0.5
1.0
1.5
2.0
2.5
1 2
∗
SNAI1
adG
FP
adSN
AI1
α-tubulin
0
2
4
6
8
10
12
14
0 12 24 36 48 60 72 84
–– +
PLEKHA7 shRNA – + +
No. 8NT No. 10
Figure 3 PLEKHA7 suppresses anchorage-independent growth and
expression of transformation-related markers. (a) Caco2 non-target control
shRNA (NT) and PLEKHA7 shRNA knockdown cells were measured for
cell impedance using the ECIS apparatus to assess junctional resistance
(mean ± s.d. from n=3 independent experiments calculated for 96 h and
∼7,000 continuous time points from 40,000 cells per condition, NT or
PLEKHA7 shRNA, per experiment; ∗
P <0.03 is shown for every 12 h interval,
Student’s two-tailed t-test). (b) Caco2 control (NT) and PLEKHA7 knockdown
(PLEKHA7 shRNA) cells co-stained for PLEKHA7 and actin (phalloidin) and
quantified (right panel) for apical ring phalloidin intensity (mean ± s.d. from
n = 3 independent experiments, assessing 40 cells in total per condition,
NT or PLEKHA7 shRNA, ∗
P =0.01, Student’s two-tailed t-test; PLEKHA7
background staining is an artefact of paraformaldehyde fixation for actin).
Scale bar for full images, 20 µm; for enlarged parts, 10 µm. (c) Caco2 control
(NT) and PLEKHA7 knockdown cells using two shRNAs (PLEKHA7 shRNA
no. 8, no. 10) were quantified for colony formation on soft agar (mean ± s.d.
from n=3 independent experiments; ∗
P <0.003, Student’s two-tailed t-test;
see Supplementary Fig. 3c for representative scan). (d–f) Caco2 control
(NT) and PLEKHA7 knockdown cells using two shRNAs (no. 8, no. 10)
analysed by western blot for the indicated markers. Phosphorylation sites are
denoted by p-. Actin is the loading control. (g) Western blot of PLEKHA7
knockdown Caco2 cells (PLEKHA7 shRNA) after ectopic re-expression of
PLEKHA7 (LZRS-PLEKHA7) for the markers shown. Actin is the loading
control. (h) Western blot and soft agar assay quantification of Caco2 cells
infected with either vector control (adGFP) or a SNAI1-expressing construct
(adSNAI1) (mean ± s.d. from n = 3 independent experiments; ∗
P = 0.006
Student’s two-tailed t-test; see Supplementary Fig. 3e for representative
scan). α-tubulin is the loading control. Source data for a–c,h are provided
in Supplementary Table 2. See Supplementary Fig. 8 for unprocessed blot
scans of d–g.
in normal and cancer samples of breast and kidney revealed that
PLEKHA7 is either mislocalized or lost in the tumour tissues, although
p120 and E-cadherin are still expressed and junctional to a high
percentage in the same samples (Supplementary Fig. 4a,b). These
data indicate the selective disruption of the ZA in tumour tissues, in
agreement with our in vitro findings.

ARTICLES
The basolateral complex promotes cell growth and expression
of related markers
Next, we examined whether the events induced by disruption of the
apical complex are due to the activity of the basolateral complex.
In agreement, both the increased growth and the elevated levels
of transformation-related markers in PLEKHA7-depleted cells were
abolished when p120 was simultaneously knocked down (Fig. 4a,b
and Supplementary Fig. 4c). PLEKHA7 knockdown did not affect
E-cadherin levels; however, p120 depletion reduced E-cadherin levels,
as expected2,5
(Supplementary Fig. 4d). Interestingly, simultaneous
knockdown of E-cadherin decreased the levels of SNAI1, CCND1,
MYC and phosphorylation of p130CAS in PLEKHA7-depleted cells
(Fig. 4c), indicating that the effects observed in the absence of
PLEKHA7 depend on E-cadherin-based junctions. Furthermore,
suppression of Src activity using the PP2 inhibitor abolished p120
and p130CAS phosphorylation and also reversed the increased
expression of SNAI1, CCND1, MYC and cadherin 11 after PLEKHA7
knockdown (Fig. 4d and Supplementary Fig. 4e). Re-expression of
wild-type murine p120 (mp120-1A; ref. 25) in p120 knockdown
cells partially restored p120 phosphorylation as well as SNAI1 and
cadherin 11 levels, whereas expression of a non-phosphorylatable
p120 construct (mp120-8F; ref. 25) at similar levels failed to reproduce
these effects (Fig. 4e).
Depletion of cadherin 11, which was previously implicated in
p120-mediated growth and migration signalling14,15
, also reversed the
increased levels of SNAI1, CCND1 and MYC on PLEKHA7 depletion
(Fig. 4f). As E-cadherin levels are unaltered in the PLEKHA7-depleted
cells (Fig. 4c and Supplementary Fig. 4d), the data do not suggest a
cadherin switch, but rather that the effects of PLEKHA7 depletion are
mediated by the cadherin-based basolateral junctions. Indeed, p120
tyrosine phosphorylation occurs only at the junctions (Fig. 1b and
Supplementary Figs 1e,f and 4e; refs 32,33), and cadherin 11 exhibits
strong junctional staining after PLEKHA7 knockdown (Fig. 4g). Col-
lectively, the results indicate that the basolateral complex acts through
E-cadherin, cadherin 11, Src and p120 to promote transformation-
related signalling, after disruption of the apical complex.
PLEKHA7 suppresses expression of SNAI1, MYC and CCND1
through miRNAs
PLEKHA7 knockdown did not significantly alter the messenger RNA
(mRNA) levels of the examined markers (Supplementary Fig. 5a),
did not prolong protein stability after cycloheximide treatment
(Supplementary Fig. 5b) and did not alter the rate of stabilization after
MG-132 treatment (Supplementary Fig. 5c). PLEKHA7 knockdown
also did not affect the phosphorylation of GSK3B, a dominant
regulator of SNAI1 stability34,35
(Supplementary Fig. 5d). These
results suggest that PLEKHA7 is not affecting the examined markers
transcriptionally or post-translationally, but imply that it may suppress
mRNA expression. A core such mechanism is RNA interference
(RNAi), which involves the miRNA-mediated silencing of mRNA
expression36–39
and is commonly deregulated in cancer40,41
. To
examine this, we used the NanoString platform and found significant
changes in the levels of 29 miRNAs out of 735 examined in PLEKHA7-
depleted cells: 15 of them were decreased and 14 miRNAs increased
(Fig. 5a). The strongest decrease was observed for miR-24, miR-30a
and let-7g, and the strongest increase for miR-19a. We also focused on
the most abundant of the affected miRNAs, namely miR-30b, which
was among those decreased (Fig. 5a). Quantitative PCRs with reverse
transcription (qRT–PCRs) confirmed the NanoString results (Fig. 5b),
whereas PLEKHA7 re-expression rescued the altered miRNA levels
(Supplementary Fig. 5e,f).
We then used anti-miRNAs to target miR-24, miR-30a, miR-30b
and let-7g in wild-type PLEKHA7-containing cells, to mimic the
effect of PLEKHA7 knockdown on their expression. Indeed, the anti-
miRNA transfections resulted in increased levels of SNAI1, MYC and
CCND1 (Fig. 5c), similar to the effect of PLEKHA7 depletion (Fig. 3f).
The miR-30 and let-7 miRNA families are validated suppressors of
SNAI1 (refs 42–44), whereas miR-24 and the let-7 family are also
verified suppressors of MYC (refs 45–47). Re-expression of miR-30b in
PLEKHA7-depleted cells using a miR-30b mimic construct reversed
the increase of SNAI1, MYC and CCND1 (Fig. 5d), confirming that
miR-30b is a mediator of PLEKHA7’s growth-suppressing signalling.
Interestingly, anti-miR-30a and anti-miR-30b also increased Src and
p120 phosphorylation, whereas the miR-30b mimic reversed their
increase in PLEKHA7-depleted cells (Supplementary Fig. 5g,h). In
addition, ectopic SNAI1 overexpression (Fig. 3h) induced p120
phosphorylation at Tyr 228 (Supplementary Fig. 5i), suggesting that
the effect of miR-30b on p120 phosphorylation is mediated by SNAI1.
In summary the data reveal that a specific subset of miRNAs, and in
particular miR-30b, are regulated by PLEKHA7 and mediate its effects
on proteins involved in transformed cell growth.
The ZA associates with the microprocessor complex
Although PLEKHA7 knockdown decreased levels of mature miR-30b
(Fig. 5a,b), it did not affect expression of the primary miR-30b
(pri-miR-30b) transcript (Fig. 6a); however, it resulted in decreased
levels of the next precursor, pre-miR-30b (Fig. 6a and Supplementary
Fig. 6a). This suggested that PLEKHA7 affects pri-miR-30b processing
to pre-miR-30b, a step in miRNA biogenesis catalysed by the
microprocessor complex and its two essential components DROSHA
and DGCR8 (refs 48–51). We tested whether PLEKHA7 depletion
affects either the total levels of DROSHA or DGCR8, or their
localization, because the microprocessor is thought to function in
the nucleus. Whereas both the overall levels of the two proteins and
their subcellular distribution remained unchanged, cell fractionation
assays revealed the existence of a non-nuclear fraction of both
DROSHA and DGCR8 (Fig. 6b). IF and confocal microscopy
confirmed their presence outside the nucleus, particularly at areas
of cell–cell contact (Fig. 6c), and revealed their co-localization with
PLEKHA7 and p120 strictly at the apical ZA of polarized epithelial
cells (Fig. 6d–g and Supplementary Fig. 6b–e). The use of alternative
antibodies for DROSHA and DGCR8 (Supplementary Fig. 6b,c)
as well as elimination of their staining after knockdown by short
interfering RNAs (siRNAs) (Supplementary Fig. 6f,g) confirmed the
specificity of their junctional localization. In agreement, DROSHA
and DGCR8 co-precipitated with PLEKHA7 (Fig. 6h,i), p120 (Fig. 6i
and Supplementary Fig. 6h) and E-cadherin (Supplementary Fig. 6h).
Notably, the interaction of PLEKHA7 with the microprocessor is RNA
independent (Fig. 6j), excluding the possibility that these interactions
are due to non-specific isolation of large ribonucleoprotein granules
from the cells. Proximity ligation assay confirmed that the interaction
of DROSHA and PLEKHA7 occurs only at the junctions (Fig. 6k).

ARTICLES
0.5
1.0
1.5
2.0
1 2 3
Foldchange
Cad11
Actin
PLEKHA7
a
e f g
b c d
CCND1
SNAI1
p120
p120 shRNA
PLEKHA7 shRNA
MYC
∗
p120 shRNA –– +
p-p120: Y228
SNAI1
Actin
p120
N
T
Vector
m
p120-1A
m
p120-8F
p120
shRN
A
Cad11
SNAI1
CCND1
p120
p-p120: Y228
MYC
Actin
Src inh: PP2
PLEKHA7 shRNA – –
–
+
+
+
–+
PLEKHA7
Cad11
SNAI1
Actin
Cad11 shRNA
CCND1
MYC
PLEKHA7
PLEKHA7 shRNA
Cad11
Ecad
SNAI1
MYC
CCND1
Actin
Ecad shRNA
PLEKHA7 shRNA
PLEKHA7
p130CAS
p-p130CAS: Y165
p130CAS
p-p130CAS: Y165
p130CAS
p-p130CAS: Y165
– + +PLEKHA7 shRNA
– + +
–– +
– + +
–– +
– – + +
– +–+
Cad11PLEKHA7
PLEKHA7 shRNA
NT
Figure 4 The basolateral junctional complex promotes anchorage-
independent growth and expression of transformation-related markers.
(a) Caco2 control, PLEKHA7 knockdown (PLEKHA7 shRNA) and PLEKHA7–
p120 double knockdown (PLEKHA7 shRNA, p120 shRNA) cells were
grown on soft agar and quantified for colony formation (mean ± s.d.
from n = 3 independent experiments; ∗
P = 0.0008, Student’s two-tailed
t-test; see Supplementary Fig. 4c for representative scan). Source
data are provided in Supplementary Table 2. (b) Control, PLEKHA7
knockdown (PLEKHA7 shRNA) and PLEKHA7–p120 double knockdown
(PLEKHA7 shRNA, p120 shRNA) Caco2 cells were analysed by western blot
for the markers shown. Actin is the loading control (see also Supplementary
Fig. 4d). (c) Control, PLEKHA7 knockdown (PLEKHA7 shRNA) and
PLEKHA7/E-cadherin (PLEKHA7 shRNA, Ecad shRNA) double knockdown
Caco2 cells were analysed by western blot for the markers shown. Actin
is the loading control. (d) Western blot of Caco2 control or PLEKHA7
knockdown (PLEKHA7 shRNA) cells after treatment with vehicle control
(dimethylsulphoxide, DMSO) or 10 µM Src inhibitor PP2; actin is the
loading control (see also Supplementary Fig. 4e). (e) Western blot of p120
knockdown Caco2 cells (p120 shRNA) after ectopic expression of either
vector control (vector), murine full-length (isoform 1A) wild-type p120
(mp120-1A) or murine full-length non-phosphorylatable p120 (mp120-
8F) for the markers shown. Actin is the loading control. (f) Western blot
of single and double PLEKHA7 (PLEKHA7 shRNA) and cadherin 11
(Cad11 shRNA) knockdown Caco2 cells for the markers shown. Actin
is the loading control. (g) IF of control (NT) or PLEKHA7 knockdown
(PLEKHA7 shRNA) cells for PLEKHA7 and cadherin 11 (Cad11). Scale
bar: 20 µm. See Supplementary Fig. 8 for unprocessed blot scans
of b–f.
The junctional localization of DROSHA and DGCR8 was disrupted
in PLEKHA7-depleted cells, indicating that it is PLEKHA7 dependent
(Fig. 7a,b). Removal of PLEKHA7 from the junctions by the
mp120- ARM1 mutant also resulted in compromised junctional
localization of DROSHA and DGCR8 (Supplementary Fig. 7a–c).
Furthermore, depletion of p120, which is required for PLEKHA7
recruitment to the junctions (Supplementary Fig. 2c), also negatively
affected DROSHA junctional localization (Supplementary Fig. 7d)
and decreased the levels of the examined miRNAs (Supplementary
Fig. 7e), mimicking the effects of PLEKHA7 depletion. Conversely,
strengthening of the junctions by Src inhibition33
increased junctional
localization of PLEKHA7, DROSHA and DGCR8 (Fig. 7c–e). As
PLEKHA7 tethers the ZA to the microtubules21
, we examined
whether the localization of DROSHA and DGCR8 at the ZA
is microtubule dependent. Calcium switch assays showed that
the junctional localization of PLEKHA7, DROSHA and DGCR8
recovers rapidly after re-addition of calcium, and is not affected
by dissolution of microtubules by nocodazole (Fig. 7f–i). Similarly,
neither prolonged nocodazole treatment (Supplementary Fig. 7f), nor
NEZHA knockdown (Supplementary Fig. 7g,h) affected the junctional
localization of PLEKHA7, DROSHA or DGCR8. Although both
miR-30b (Fig. 5d) and p120 or cadherin 11 knockdown (Fig. 4b,f)
rescue the levels of pro-tumorigenic markers in PLEKHA7-depleted
cells, simultaneous knockdown of either p120 or cadherin 11 with
PLEKHA7 did not reverse either the decreased miRNA levels
(Supplementary Fig. 7i) or the compromised junctional localization
of DROSHA and DGCR8 (Supplementary Fig. 7j–m), indicating that
these events are PLEKHA7 specific and that p120 and cadherin 11
induce cell transforming markers through a separate pathway. Taken
together, the data reveal the association of the microprocessor complex
with the apical ZA in non-transformed epithelial cells, in a PLEKHA7-
dependent manner.

ARTICLES
NT
PLEKHA7 shRNA no. 8
PLEKHA7 shRNA no. 10
∗
∗
∗
∗
∗
∗
∗
∗
hsa-m
iR-24
hsa-m
iR-30ahsa-let-7g
hsa-m
iR-183hsa-let-7i
hsa-m
iR-30d
hsa-m
iR-450a
hsa-m
iR-148b
hsa-m
iR-301a
hsa-m
iR-30b
hsa-m
iR-30e
hsa-m
iR-15b
hsa-m
iR-103
hsa-m
iR-221
hsa-m
iR-203
hsa-m
iR-450b-5p
hsa-m
iR-296-5p
hsa-m
iR-223
hsa-m
iR-130b
hsa-m
iR-423-3p
hsa-m
iR-619
hsa-m
iR-1274b
hsa-m
iR-934
hsa-m
iR-1246
hsa-m
iR-331-3p
hsa-m
iR-302b
hsa-m
iR-302a
hsa-m
iR-302d
hsa-m
iR-19a
Foldchange(P<0.05)
Foldchange NanoString: PLEKHA7 shRNA versus NT
Relative abundance
Relative abundance
∗
∗
SNAI1
C
ontrola-m
iR-24a-m
iR-30aa-m
iR-30b
C
ontrol
C
ontrolm
iR-30b
m
iR-30b
a-let-7g
Actin
CCND1
MYC
SNAI1
Actin
CCND1
MYC
NT PLEKHA7 shRNA
PLEKHA7
–3
–2
–1
0
1
2
3a
b d
c
2331
121
291
852
599
32
161
171
35
193
108
146
554
196
344
110
24
29
16
38
34
21
94
36
272
46
39
61
31
0
m
iR-24
m
iR-30a
m
iR-30b
m
iR-19a
let-7g
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Figure 5 PLEKHA7 suppresses protein expression through miRNAs. (a) Caco2
control (NT) and PLEKHA7 knockdown (PLEKHA7 shRNA) cells analysed
for miRNA expression using NanoString. Results shown are statistically
signiﬁcant changes (mean ± s.d. from n = 3 independent experiments;
P < 0.05, Student’s two-tailed t-test) shown as average fold change of
PLEKHA7 shRNA versus control cells. Numbers on each bar are NanoString
counts indicating relative abundance of the respected miRNAs (see also
Methods). Source data are deposited at Gene Expression Omnibus, accession
number GSE61593. (b) qRT–PCR analysis of Caco2 control (NT) or PLEKHA7
knockdown (PLEKHA7 shRNA no. 8, no. 10) cells for the indicated miRNAs
(mean ± s.d. from n = 3 independent experiments; ∗
P < 0.01, Student’s
two-tailed t-test). Source data are provided in Supplementary Table 2.
(c) Western blot of Caco2 cells transfected with the indicated anti-miRNAs
for the markers shown; actin is the loading control. (d) Caco2 control (NT)
and PLEKHA7 knockdown (PLEKHA7 shRNA) cells were transfected with
either control or miR-30b mimic and blotted for the markers shown; actin
is the loading control. See Supplementary Fig. 8 for unprocessed blot scans
of b–d.
PLEKHA7 regulates processing of pri-miR-30b at the junctions
Subcellular fractionation revealed the existence of at least three
pri-miRNAs, pri-miR-30b, pri-let-7g and pri-miR-19a, outside the
nucleus (Fig. 8a), and RNA-IPs indicated that PLEKHA7 is enriched
in these pri-miRNAs (Fig. 8b). In vitro processing assays using
pri-miR-30b as a template showed that the PLEKHA7-associated
complex possesses pri-miRNA processing activity (Fig. 8c). PLEKHA7
depletion decreased in vitro processing of pri-miR-30b by about 30%
(Fig. 8d,e), similar to the decrease observed in endogenous pri-miR-
30b processing after PLEKHA7 knockdown (Fig. 6a). Finally, in situ
hybridization (ISH) confirmed the localization of pri-miR-30b at
the junctions (Fig. 8f). Collectively, our results demonstrate that the
ZA-localized microprocessor complex is active and that PLEKHA7
regulates processing at least of pri-miR-30b at the ZA.
In summary, the data support a model whereby the apical ZA
complex recruits the microprocessor through PLEKHA7 to suppress
growth-related signalling promoted by the basolateral junctional
complex, hence maintaining the normal epithelial architecture and
behaviour (Fig. 8g).
DISCUSSION
Our work shows that the dual p120 and E-cadherin behaviour
in epithelial cell growth can be explained by the existence of
two distinct p120 complexes with opposing functions: an apical
complex at the ZA that suppresses growth-related signalling
through PLEKHA7-mediated regulation of select miRNAs and
a basolateral complex that promotes anchorage-independent
growth and increased expression of markers such as SNAI1, MYC

ARTICLES
DROSHA
Non-polarized
DROSHAPLEKHA7
ApicalBasal
x
z
DGCR8PLEKHA7
x
y
x
z
x
y
x
z
x
DROSHA
p120
DGCR8
Proxim
ity
ligation
Phase–DAPI–
proxim
ity
ligation
p120
PLEKHA7
p120
DROSHA
WBs
DGCR8
Input
IgG
PLEKH
A7p120
IPs
DROSHA
DGCR8
PLEKHA7
p120
WBs
Input
Input
IgG
IgGDrosha
PLEKH
A7
PLEKH
A7
+
RN
Ase
DG
C
R8
IPs
ApicalBasal
DAPI
DAPI
DAPI
Merged
Merged
PLEKHA7+
DROSHA
PLEKHA7+
p120 Neg. control
DGCR8
0
pri-m
iR-30b
pre-m
iR-30b
0.2
0.4
0.6
0.8
1.0
1.2
∗Foldchange
NT
PLEKHA7 shRNA no. 8
a b
d
e
f g
c
h
i
j
k
Histone H3
DROSHA
DGCR8
Lamin A/C
GAPDH
NuclearTotal
Actin
Non-
nuclear
Merged
PLEKHA7
DROSHA
WBs
DGCR8
IPs
z
N
T
PLEKH
A7
shRN
A
PLEKH
A7
shRN
A
N
T
N
TPLEKH
A7
shRN
A
Figure 6 PLEKHA7 associates with DROSHA and DGCR8 at the ZA.
(a) qRT–PCR analysis of pri-miR-30b and pre-miR-30b in Caco2 control (NT)
or PLEKHA7 knockdown (PLEKHA7 shRNA) cells (mean ± s.d. from n=3
independent experiments; ∗
P = 0.02, Student’s two-tailed t-test). Source
data are provided in Supplementary Table 2. (b) Western blot of either
total cell lysates or of nuclear and non-nuclear lysates after subcellular
fractionation of Caco2 NT or PLEKHA7 shRNA cells for the indicated
markers. Lamin A/C and histone H3 are the nuclear and GAPDH the
cytoplasmic markers; actin is the total loading control. (c) IF of non-
polarized Caco2 cells, co-stained for DROSHA and DGCR8; 4,6-diamidino-
2-phenylindole (DAPI) is the nuclear stain. (d,e) IF of polarized Caco2
cells co-stained for PLEKHA7 and DROSHA or DGCR8. Enlarged details in
boxes are shown above the apical ﬁelds; a composite x–z merged image is
shown at the side of the x–y image stack (antibodies used here: DROSHA,
Abcam; DGCR8, Abcam; see also Supplementary Fig. 6b–e for a second
set of antibodies used and Supplementary Table 3 for antibody details).
DAPI is the nuclear stain. (f,g) Composite x–z images of polarized Caco2
cells co-stained by IF for p120 and DROSHA or DGCR8 (see Supplementary
Fig. 6b,c, respectively, for x–y set of image stacks). (h,i) Western blots of
PLEKHA7, p120, DROSHA and DGCR8 IPs for the markers shown. IgG is
the negative IP control. (j) Western blot of PLEKHA7 IPs with and without
RNAse treatment; IgG is the negative control. (k) Proximity ligation assay
using PLEKHA7 and DROSHA antibodies, PLEKHA7 and p120 antibodies
(assay positive control) or a p120 antibody only (assay negative control); the
composite phase contrast–DAPI–proximity ligation images are also shown.
Scale bars for x–y images, 20 µm; for x–z images, 5 µm; for enlarged parts
of d and e, 3 µm. See Supplementary Fig. 8 for unprocessed blot scans
of b,h–j.

ARTICLES
PLEKHA7 shRNA
NT
DROSHA DGCR8p120
PP2
Control
PLEKHA7p120
PP2
Control
DROSHA DGCR8
α-tubulin PLEKHA7
Nocodazole
Control Control
Control
Nocodazole
Nocodazole
Ca2+: 0 min Ca2+: 30 min Ca2+: 60 min
Ca2+: 0 min
Control
Nocodazole
Ca2+: 30 min Ca2+: 60 min
p120DROSHAa b
c
f g
h i
d e
PP2
Control
p120
PLEKHA7 shRNA
NT
PLEKHA7 DGCR8
Figure 7 Localization of DROSHA and DGCR8 at the ZA is PLEKHA7
dependent. (a,b) IF of control (NT) or PLEKHA7 knockdown
(PLEKHA7 shRNA) Caco2 cells for DROSHA and DGCR8, co-stained for
PLEKHA7 or p120. PLEKHA7 background intracellular staining in a is an
artefact of paraformaldehyde fixation. (c–e) Caco2 cells treated either with
vehicle control (DMSO) or the Src inhibitor PP2 (10 µM) were stained by IF
for PLEKHA7, DROSHA and DGCR8 and co-stained with p120. (f–i) Calcium
switch assay of Caco2 cells for the indicated times after Ca2+
re-addition
(Ca2+
: 0 min indicates Ca2+
depleted cells immediately before Ca2+
re-
addition), treated with vehicle (control) or with 10 µM nocodazole, and
stained by IF for α-tubulin, PLEKHA7, DROSHA and DGCR8. Enlarged
image details are shown in yellow boxes on the right-hand side of the image
stacks in a, b, d and e. Scale bars for full images, 20 µm; for enlarged
parts, 8 µm.
and CCND1 through activated Src and tyrosine-phosphorylated
p120 (Fig. 8g).
DROSHA and DGCR8 are thought to regulate miRNA processing
in the nucleus48–50
. However, these proteins have not been studied
in the context of non-transformed, polarized mammalian epithelial
cells. Our data reveal that, in these cells, a functional subset of the
microprocessor is recruited by PLEKHA7 to the ZA (Figs 6d–k and
7a,b). PLEKHA7 does not globally regulate miRNA expression in
the cells examined, but affects only a specific subset of miRNAs
(29 out of 735 examined; Fig. 5a) in a confluence-independent
manner, because all experiments herein were carried out in fully
confluent epithelial monolayers. Importantly, the interaction of
PLEKHA7 with DROSHA (Fig. 6k) or pri-miR-30b (Fig. 8b,f)
occurs exclusively at the ZA, whereas depletion of PLEKHA7
dissociates DROSHA and DGCR8 from the ZA and reduces miR-
30b processing, without affecting nuclear DROSHA and DGCR8
levels (Figs 6b, 7a,b and 8d,e). Therefore, the present study does
not dispute the general model of miRNA biogenesis, but uncovers
a mechanism whereby PLEKHA7 recruits a subpopulation of the
microprocessor to the ZA and regulates processing of select miRNAs
to suppress the expression of markers involved in transformed
cell growth.

ARTICLES
pri-
miR-30b
pre-
miR-30b
1,000
21
50
80
150
300
500
nt:
IPs:
IgG
Low exposure
pri-
miR-30b
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Foldenrichment(log10)
pri-miR-30b
pri-let-7g
pri-miR-19a
a
c
f g
d
e
b
Nuclear
Non-nuclear
NT PLEKHA7 shRNA
IgG
PLEKH
A7
DG
C
R8
PLEKH
A7
DG
C
R8
IgG
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DG
C
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PLEKH
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miR-30b
1,000
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nt:
IPs:
0
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Foldchange
Relative
pre-miR-30b
NT PLEKHA7
shRNA
∗
Ecad
pri-miR-30b
DAPI !
Ecad
DapB
DAPI
Ecad
PPIB
DAPI
Positive cont.Negative cont.
Histone H3
DROSHA
DGCR8
Lamin A/C
GAPDHN
on-nuclear
pri-m
iR-30b
pri-let-7g
pri-m
iR-19a
AC
TB
N
uclear
ZA
Apical
Basal
Lateral
PLEKHA7
DROSHA–DGCR8
p130CAS
p190RhoGAP
p-Src
SNAI1
MYC
CCND1
p120–Ecad
p120–Cad11
AIGp-p120
miR-30b
ZA
pri-miR-30b
p120–Ecad
18
20
22
24
26
28
30
32
34
36
38
16
40
qPCR:Ctvalues
Figure 8 PLEKHA7 regulates pri-miR-30b processing at the junctions.
(a) qRT–PCR of isolated nuclear and non-nuclear subcellular fractions
of Caco2 cells for the indicated pri-miRNAs (right panel). Successful
fractionation is demonstrated by western blot for the indicated markers
(left panel). Ct values are shown (mean ± s.d. from n = 3 independent
experiments); actin (ACTB) is the positive qRT–PCR control. Lamin A/C and
histone H3 are the nuclear and GAPDH the cytoplasmic markers for the
western blot. See Supplementary Fig. 8 for unprocessed blot scans. (b) qRT–
PCR of the eluates from the IgG (negative control), PLEKHA7 and DGCR8
(positive control) RNA-IPs for the indicated pri-miRNAs (a representative
is shown from two independent experiments). (c) In vitro pri-miR-30b
processing activity assay of Caco2 lysates for PLEKHA7, DGCR8 (positive
control) and IgG (negative control) IPs. A lower exposure image of the pri-miR-
30b template is presented to show the actual loading amount. nt, nucleotides.
(d) In vitro pri-miR-30b processing activity assay for DGCR8 IPs of NT or
PLEKHA7 shRNA Caco2 cells; IgG is the negative control. (e) Quantification
of pre-miR-30b band intensities from n=3 independent in vitro processing
activity assays, carried out as in d (mean ± s.d.; ∗
P <0.001, Student’s two-
tailed t-test). (f) ISH of Caco2 cells for pri-miR-30b; a bacterial DapB RNA
probe was used as the negative and the endoplasmic-reticulum-specific PPIB
as the positive control. Merged images are shown. An enlarged area in the
yellow box in the top right panel is shown for the pri-miR-30b ISH. Arrows
indicate hybridization signals. Scale bars for full images, 20 µm; for enlarged
part, 12 µm. Source data are provided in Supplementary Table 2 for a, b and
e. (g) Schematic diagram summarizing the findings of the present study. Two
distinct junctional complexes exist in polarized epithelial cells: a basolateral
complex lacking PLEKHA7 promotes growth signalling and anchorage-
independent growth (AIG) through cadherins, Src and p120 activity, whereas
an apical ZA complex suppresses these events through PLEKHA7, by locally
recruiting the microprocessor at the ZA to regulate processing of miR-30b.

ARTICLES
The miRNAs regulated by PLEKHA7 provide a mechanistic
link to the essential function of the ZA in maintaining epithelial
homeostasis. The miR-30 and let-7 families are well established
guardians of the epithelial phenotype and epithelial–mesenchymal
transition suppressors42–44
. In contrast, the most strongly increased
miR-19a after PLEKHA7 depletion is a key component of the
polycistronic miR-17-92 miRNA oncogene52,53
. Our data support a
model whereby individual cells within an epithelial monolayer sense
the status of their apical ZA by regulating the levels of a subset of
miRNAs to accordingly suppress or promote growth (Fig. 8g).
Co-localization of phosphorylated p120 with activated Src and
Rac1 was observed specifically at the basolateral complex, similar
to the case of p190RhoGAP, which suppresses RhoA signalling and
induces transformed cell growth in a Rac1- and Src-dependent
manner7,13
(Figs 1b,d,g,h, 2d, and Supplementary Fig. 1e,f,h,i).
Therefore, it is the basolateral complex that exerts the previously
described Src- and Rac1-induced transforming activity of p120
(refs 13,15). In agreement, we show that E-cadherin, Src activation
and p120 tyrosine phosphorylation are required for increased growth
signalling in the absence of PLEKHA7 (Fig. 4b–e). Therefore,
the growth-suppressing or growth-promoting functions of p120
and E-cadherin are fine-tuned by binding to PLEKHA7. Further
interactions of PLEKHA7 with paracingulin54
, afadin55
, ZO-1 and
cingulin56
, may also play a role in PLEKHA7 function, although the
interaction with p120 is essential for modulating cell behaviour by
regulating miRNA processing.
Taken together, our data untangle the complicated roles of
E-cadherin and p120 in the context of distinct junctional complexes,
spatially separating their functions and providing an explanation for
their conflicting behaviour in cell growth. In addition, they identify
PLEKHA7 as a specific marker of ZA that mediates suppression of
growth-related signalling. Finally, they reveal an interaction of the ZA
with the microprocessor complex, and uncover a mechanism whereby
the ZA regulates a set of miRNAs to suppress cellular transformation
and maintain the epithelial phenotype.
METHODS
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary Information is available in the online version of the paper
ACKNOWLEDGEMENTS
This work was supported by NIH R01 CA100467, R01 NS069753, P50 CA116201
(P.Z.A.); NIH R01 GM086435, Florida Department of Health, Bankhead-Coley
10BG11 (P.S.); NIH/NCI R01CA104505, R01CA136665 (J.A.C.); BCRF (E.A.P.); the
Swiss Cancer League (S.C., Project KLS-2878-02-2012). A.K. is supported by the Jay
and Deanie Stein Career Development Award for Cancer Research at Mayo Clinic.
We thank Mayo Clinic’s Proteomics Core and B. Madden for assistance with mass
spectrometry, B. Edenfield for immunohistochemistry, M. Takeichi, D. Radisky and
M. Cichon for constructs, and D. Radisky, L. Lewis-Tuffin, J. C. Dachsel, B. Necela
and the late G. Hayes for suggestions and comments.
AUTHOR CONTRIBUTIONS
A.K. designed the study, conceived and designed experiments, carried out all
experiments except those described below, analysed the data and wrote the
manuscript. S.P.N. made constructs. P.P. and S.C. developed antibodies and
constructs. R.W.F. provided technical support. L.R.C. assisted with IF. T.R.B., J.M.C.
and E.A.T. carried out the NanoString experiment and assisted with qRT–PCRs.
I.K.Y. and T.P. assisted with the ISH assay. E.A.P., P.S., S.B. and J.A.C. developed and
provided tissue micro-arrays. P.Z.A. conceived and designed the study, conceived
and designed experiments and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://dx.doi.org/10.1038/ncb3227
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METHODS DOI: 10.1038/ncb3227
METHODS
Cell culture, reagents and chemicals. In all comparisons, cells were used strictly at
the same confluences. All cell lines were obtained from ATCC, used at low passage
(<20), and tested negative for mycoplasma contamination. Caco2 colon epithelial
cells were cultured in MEM (Cellgro) supplemented with 10% FBS (Invitrogen),
1 mM sodium pyruvate (Invitrogen) and 1× non-essential amino-acid supplement
(Mediatech). MDCK canine kidney epithelial cells, HEK 293FT human embryonic
kidney cells, and Phoenix-Ampho cells were cultured in DMEM supplemented
with heat-inactivated 10% FBS. PP2 was obtained from Calbiochem; nocodazole,
cycloheximide and MG-132 from Sigma.
Constructs. Full-length human PLEKHA7 was cloned in the XbaI–HindII sites
of pcDNA3.1myc–His and then the PLEKHA7–Myc fragment was cut out by
PmeI and sub-cloned to the AfeI site of the retroviral LZRS vector to obtain the
LZRS-PLEKHA7–Myc construct. The PLEKHA7–GFP construct was a gift of the
M. Takeichi laboratory (RIKEN, Japan; ref. 21). The mp120-1A, mp120-4A, mp120-
8F and mp120- ARM1 constructs have been previously described2,25
. The adSNAI1
and adGFP adenoviruses were a gift of the D. Radisky laboratory (Mayo Clinic, FL,
USA). The YFP–PBD (plasmid 11407) and GFP–rGBD (plasmid 26732) constructs
were obtained from Addgene.
shRNAs, siRNAs, anti-miRNAs and miR-mimics. Cells were transfected using
Lipofectamine 2000 (Invitrogen) or Lipofectamine RNAiMAX (Invitrogen) for
anti-miRNA, miR-mimic and siRNA transfection, according to the manufacturer’s
protocols. Lentiviral shRNAs were derived from the pLKO.1-based TRC1
(Sigma–RNAi Consortium) shRNA library (pLKO.1-puro non-target shRNA
control, SHC016; PLEKHA7 no. 8, TRCN0000146289; PLEKHA7 no. 10,
TRCN0000127584; Cad11, TRCN0000054334; Ecad no. 21, TRCN0000039664;
Ecad no. 23, TRCN0000039667) and the pLKO.5-based TRC2 library (pLKO.5-puro
non-target shRNA control, SHC216; NEZHA no. 72, TRCN0000268676; NEZHA,
no. 73: TRCN0000283758). Lentiviruses were produced in HEK 293FT cells and
used to infect cells according to standard protocols. Retroviruses were prepared in
Phoenix-Ampho cells, as described previously15
. SMARTpool (Dharmacon) siRNAs
used: DROSHA, M-016996-02-0005; DGCR8, M-015713-01-0005; non-target,
D-001206-14-05 mirVana (Life Technologies catalogue no. 4464084). Anti-miRNAs
used: anti-hsa-miR-30a, ID MH11062; anti-hsa-let-7g, ID MH11758; anti-hsa-
miR-30b, ID MH10986; anti-hsa-miR-24, ID MH10737; negative control no. 1,
catalogue no. 4464076. MISSION (Sigma) miR-mimics used: miR-30b, catalogue
no. HMI0456; negative control 1 (catalogue no. HMC0002).
Antibodies. For detailed information on all the antibodies used in the study, as well
as working dilutions for each assay, see Supplementary Table 3.
Immunofluorescence. MDCK and Caco2 cells were grown on Transwell inserts
(Costar 3413) for 7 or 21 days respectively, until they polarized, or on sterile glass
coverslips until they reached full confluence. Cells were washed once with PBS
and then fixed with either 100% methanol (Fisher) for 7 min; or 4% formaldehyde
(EMS) for 20 min, followed by 0.02% Triton X-100 permeabilization for 10 min;
or 10% TCA (Sigma) for 15 min on ice, particularly for RhoA co-staining, and
permeabilized as above. Cells were blocked with either 3% non-fat milk (Carnation)
in PBS or Protein-Block reagent (Dako, X090930-2) for 30 min and stained with
primary antibodies diluted either in milk or antibody diluent (Dako, S302281-2)
for 1 h. Cells were then washed three times with PBS, stained with the fluorescent-
labelled secondary antibodies for 1 h, washed three times with PBS, co-stained with
DAPI (Sigma) to visualize the nuclei, mounted (Aqua Poly/Mount, Polysciences)
and imaged using a Zeiss LSM 510 META laser confocal microscope, under a ×63
objective, with a further ×1.6 zoom. z-stacks were acquired at 0.5 µm intervals.
Images, stacks and x–z representations of the image stacks were processed using
the Zen software (Zeiss).
Immunoblotting. Whole-cell extracts were obtained using RIPA buffer (Tris,
pH 7.4, 50 mM NaCl, 150 mM, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) supple-
mented with protease (Cocktail III, RPI) and phosphatase inhibitors (Pierce). Lysates
were homogenized through a 29 g needle and cleared by full-speed centrifugation
for 5 min. Protein extracts were mixed with LSB and separated by SDS–PAGE,
transferred to nitrocellulose membranes (Bio-Rad), blotted according to standard
protocols, detected by luminescence using ECL (GE Healthcare) and imaged using
X-ray films (Pierce). See Supplementary Fig. 8 for original scans of western blots.
Immunohistochemistry—ethics statements. Breast tissue samples were initially
collected under protocol MC0033, with the approval of the Mayo Clinic Institutional
Review Board. Written informed consent for the use of these tissues in research was
obtained from all participants. Generation of the tissue micro-arrays was carried
out under protocol 09-001642. All unique patient identifiers and confidential data
were removed and tissue samples were de-identified. The Mayo Clinic Institutional
Review Board assessed the protocol 09-001642 as minimal risk and waived the
need for further consent. Renal tissue samples were collected for the Mayo Clinic
Renal Tissue Registry under protocol 14-03, with the approval of the Mayo Clinic
Institutional Review Board. Written informed consent for the use of these tissues
in research was obtained from all participants to the Registry. Renal tissue samples
used here were obtained from the Renal Tissue Registry under the Mayo Clinic
Institutional Review Board protocol 675-05. All unique patient identifiers and
confidential data were removed and tissue samples were de-identified. The Mayo
Clinic Institutional Review Board assessed the 675-05 protocol as minimal risk
and waived the need for further consent. All data were analysed anonymously.
Paraffin-embedded tissues on slides were placed in xylene and rehydrated in a
graded ethanol series, rinsed in water, subjected to heat antigen retrieval according
to the manufacturer’s instructions (Dako), incubated with primary antibody and
finally with either anti-rabbit (for PLEKHA7) or anti-mouse (for Ecad, p120)
labelled polymer horseradish peroxidase (Dako). Slides were scanned using Aperio
ScanScope XT and viewed using Aperio ImageScope v11.1.2.752.
Junction resistance measurements. ECIS Z (Electric Cell-substrate Impedance
System, Applied Biophysics) was used to measure impedance of Caco2 cells. 4×104
cells were plated on electrode arrays (8W10E, Applied Biophysics). Measurements
were made every 180 s at the 4,000 Hz frequency that measures junctional impedance
and at 64,000 Hz that corresponds to cell density. The 4,000 Hz values were
normalized to the 64,000 Hz values to account for cell density variations.
Soft agar assay. Six-well plates (Costar 3516) were first covered with a layer of
0.75% agarose, prepared by mixing 1.5% of sterile agarose (Seakem) in a 1:1 ratio
with ×2 concentrated Caco2 culture medium made from powder MEM (Invitrogen,
catalogue no. 61100061) and double addition of each supplement (see above). The
basal layer was left to solidify for 30 min. Cells were trypsinized, counted and
resuspended in ×2 Caco2 medium. The cell suspension was mixed in a 1:1 ratio
with 0.7% sterile agarose to make a final layer of 0.35% agarose, which was added
on top of the basal layer. 5×104
Caco2 cells were seeded per well. The top layer was
left to solidify for another 30 min and was covered with 1× Caco2 culture medium.
Cultures were maintained for 4 weeks with medium renewal on top of agar every
three days until colonies were visible, and then stained with 0.02% crystal violet
(Fisher) in 20% ethanol and PBS, washed three times with distilled H2O, scanned
and counted for colonies.
Calcium switch assay. Caco2 cells were grown on coverslips until confluency, pre-
treated with either DMSO (control) or 10 µM nocodazole for 1 h to dissolve micro-
tubules, then washed three times with calcium-free PBS and incubated in calcium-
free Caco2 medium (Life, 11380-037, supplemented with glutamine, 10%FBS,
sodium pyruvate and MEM) containing 4 mM EGTA for 30 min, until cells were
rounded, while being kept in either DMSO or 10 µM nocodazole, respectively. Cells
were then washed three times with PBS, returned to regular Caco2 medium, again
with DMSO or nocodazole respectively, and fixed for IF, for the indicated times.
IPs. Cells were grown on 10 cm plates until fully confluent, washed twice with PBS
and lysed using a Triton X-100 lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM
Tris pH 7.4, 1% Triton X-100) containing twice the amount of protease (Cocktail III,
RPI) and phosphatase inhibitors (Pierce). Four 10 cm plates (∼3–4×106
cells) were
used per IP. In parallel, 4 µg of antibody or IgG control (Jackson ImmunoResearch,
011-000-003) was incubated with 40 µl Protein G Dynabeads (Invitrogen) overnight
and then washed three times with IP lysis buffer. Cell lysates were incubated with
the bead-conjugated antibodies overnight. Beads were then washed three times with
IP lysis buffer and eluted using 50 mM dithiothreitol (Sigma) in lysis buffer at 37 ◦
C
for 45 min, with constant agitation. For RNAse-treated IPs, beads were distributed
equally after the final wash into two tubes with either 1.5 ml PBS or PBS containing
100 mg ml−1
RNAse A (Sigma). After incubation at 4 ◦
C for 1.5 h, beads were washed
three times with PBS, and proteins were eluted as above.
Proteomics. Caco2 cells were grown on 10 cm Transwell inserts (Costar 3419)
for 21 days to polarize. Cells were then washed twice with PBS and proteins were
cross linked as previously described57
, using 0.75 mM of the reversible cross linker
DSP (Lomant’s reagent; Pierce) for 3 min at room temperature, which was then
neutralized using 20 mM Tris pH 7.5 for 15 min. Cells were finally lysed using
RIPA (see recipe above, immunoblotting section) containing twice the amount of
protease (Cocktail III, RPI) and phosphatase inhibitors (Pierce) and 1 mM EDTA.
Four 10 cm Transwells (∼3–4 × 106
cells) were used per IP. In parallel, 4 µg of
antibody or IgG control (Jackson ImmunoResearch, 011-000-003) was incubated
with 40 µl protein G Dynabeads (Invitrogen) overnight and cross linked to the beads
using 5 mM BS (ref. 3; Pierce) according to the manufacturer’s protocol. Cross linked
lysates were incubated with the bead-conjugated antibodies overnight. Beads were
NATURE CELL BIOLOGY

DOI: 10.1038/ncb3227 METHODS
then washed three times with lysis buffer and proteins were eluted as described
above and separated by SDS–PAGE. Gels were silver-stained (Pierce SilverSnap kit),
according to the manufacturer’s protocol. Gel slices were selected and excised using
sterile scalpels and destained, reduced and alkylated before digestion with trypsin
(Promega). Samples were analysed using nanoHPLC-electrospray tandem mass
spectrometry (nanoLC-ESI-MS/MS) in a ThermoFinnigan LTQ Orbitrap hybrid
mass spectrometer (ThermoElectron Bremen). Mascot (Matrix Sciences London)
was used to search the Swissprot database to identify isolated peptides. Common
contaminants such as trypsin, casein, keratin and microbial-specific proteins were
removed from analysis. Results were visualized using Cytoscape 2.8.2 (ref. 58).
Total RNA isolation and qRT–PCR. Cells were lysed using Trizol (Invitrogen) and
subjected to the Trizol Plus total transcriptome isolation protocol of the PureLink
RNA mini kit (Ambion—Life Technologies) specified to isolate both mRNAs and
miRNAs. Final RNA concentrations were determined using a NanoDrop spec-
trophotometer. RNA was converted to complementary DNA using a high capacity
cDNA reverse transcriptase kit (Applied Biosystems). qPCR reactions were carried
out using the TaqMan FAST Universal PCR master mix (Applied Biosystems), in a
7900 HT or ViiA 7 thermocycler (Applied Biosystems). Data were analysed using RQ
Manager (Applied Biosystems). U6 was used as a control for miRNA expression nor-
malization and GAPDH, β-actin for mRNA and pri/pre-miRNA normalization. Taq-
Man assays used for miRNAs (Applied Biosystems, catalogue no. 4427975): hsa-miR-
24, 000402; hsa-miR-30a, 000417; hsa-miR-30b, 000602; hsa-let-7g, 002282; hsa-
miR-19a, 000395; U6, 001973. TaqMan assays for miRNA precursors: pri-miR-30b,
Applied Biosystems, catalogue no. 4427012, Hs03303066_pri; pre-miR-30b, Custom
Plus TaqMan RNA assay catalogue no. 4441114, assay ID AJN1E56, context se-
quence 5 -CTGTAATACATGGATTGGCTGGGAG-3 . TaqMan assays for mRNAs
(Applied Biosystems, catalogue no. 4331182): PLEKHA7, Hs00697762_m1; SNAI1,
Hs00195591_m1; MYC, Hs00153408_m1; CCND1, Hs00277039_m1; GAPDH,
Hs99999905_m1; actin (ACTB), Hs99999903_m1.
RNA-IPs. RNA-IPs were carried out as described above for standard IPs, but
with the addition of 100 U ml−1
RNAse inhibitor (Promega) in the lysis buffer.
After the final wash, RNA was eluted from the beads using Trizol (Invitrogen)
and purified as described above. qRT–PCRs were then carried out as above and
the results were analysed using the Sigma RIP-qRT–PCR data analysis calculation
shell, associated with the Sigma Imprint RIP kit (http://www.sigmaaldrich.com/
life-science/epigenetics/imprint-rna.html and references therein).
Cell fractionation. To separate the nuclear fraction, cells were first lysed using a
buffer containing 25 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40,
protease (Cocktail III, RPI), phosphatase (Pierce) and RNAse (100 U ml−1
; Promega)
inhibitors for 10 min on ice. The lysates were centrifuged at 300g for 5 min at 4 ◦
C
to pellet the nuclei and the supernatant was carefully transferred to a fresh tube.
The nuclear pellet was lysed using RIPA (see recipe above, immunoblotting section)
with protease, phosphatase and RNAse inhibitors for 10 min on ice, homogenized
through a 29 g needle and cleared by centrifugation for 5 min at 12,000g. Half of
each lysate was used for SDS–PAGE, whereas the other half was mixed with Trizol
and used to extract total RNA and cary out qRT–PCRs, as described above.
Proximity ligation. The Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma) was
used according to the manufacturer’s instructions.
In vitro pri-miRNA processing assay. Total cDNA from Caco2 cells was obtained
as described above (qRT–PCR section) and was used to amplify the pri-miR-
30b transcript. The pri-miR-30b cDNA was then subcloned into the pBluescript
SK+ vector, downstream of the T7 promoter, between the EcoRI and XhoI sites.
Primers used: pri-30bF-XhoI, 5 -ATTAACTCGAGGTGAATGCTGTGCCTGTT
C-3 ; pri-30bR-EcoRI, 5 -ACGTTGAATTCGCCTCTGTATACTATTCTTGC-3 .
The cloned pri-miR-30b sequence is as follows (the pre-mRNA sequence is shown
in capital letters): 5 -gtgaatgctgtgcctgttctttttttcaacagagtcttacgtaaagaaccgtacaaactta
gtaaagagtttaagtcctgctttaaACCAAGTTTCAGTTCATGTAAACATCCTACACTC
AGCTGTAATACATGGATTGGCTGGGAGGTGGATGTTTACTTCAGCTGAC
TTGGAatgtcaaccaattaacattgataaaagatttggcaagaatagtatacagaggc-3 . The pri-miR-
30b construct was linearized using SmaI and in vitro transcription was carried out
using T7 polymerase (Roche) and fluorescein isothiocyanate (FITC) RNA labelling
mix (Roche), according to the manufacturer’s instructions. The FITC-labelled pri-
miR-30b was gel-purified and stored at −20 ◦
C in the dark for no more than a
week. For the in vitro processing assay, IPs on Caco2 lysates were carried out as
described above using IgG (negative control), a PLEKHA7 (Sigma) or a DGCR8
(Sigma) (positive control) antibody. For the PLEKHA7 knockdown experiment,
control (NT) or PLEKHA7 shRNA cells were used and IPs were carried out using
an IgG (negative control), or a DGCR8 (Sigma) antibody. After the final wash, the
beads were mixed with a reaction mixture containing 20 mM Tris at pH 8.0, 100 mM
KCl, 7 mM MgCl2, 20 mM creatine phosphate, 1 mM ATP, 0.2 mM phenylmethyl
sulphonyl fluoride, 5 mM dithiothreitol, 10% glycerol, 2 U µl−1
RNAse inhibitor and
3 µg FITC-labelled pri-miR-30b. The labelled probe was denatured for 2 min at 95 ◦
C
and left to gradually cool down and refold before the assay. The processing assay
was carried out for 90 min at 37 ◦
C in the dark with constant gentle rocking. RNA
was eluted with RNA elution buffer (2% SDS, 0.3 M NaOAc pH 5.2) purified with
phenol–chloroform–IAA (25:24:1) pH 5.5 (Fisher), and precipitated overnight at
−80◦
C using NaOAc 0.3 M pH 5.2, 1 µl glycogen (Invitrogen) and 4:1 100% EtOH.
The RNA was recovered by full-speed centrifugation for 30 min at 4 ◦
C, washed with
75% EtOH, resuspended in 7.5 µl DEPC–H2O, denatured in 1:1 Gel Loading Buffer
II (Life Technologies) for 5 min at 95 ◦
C, and loaded onto a 12% denaturing PAGE
RNA gel (SequaGel; National Diagnostics). The gel was scanned using a Typhoon
9400 (GE Healthcare) and quantification of bands was carried out using ImageJ.
ISH. For ISH of pri-miR-30b, Caco2 cells were grown on slides, fixed with 10%
formalin (Protocol; Fisher), dehydrated and rehydrated in a series of 50–70–
100–70–50% EtOH solutions, pretreated with protease and hybridized using the
RNAscope 2.0 HD Brown assay (Advanced Cell Diagnostics, ACD), according to
the manufacturer’s instructions. Probes used: human pri-miR-30b, ACD, 415331;
positive control probe Hs-PPIB, ACD, 313906; negative control probe DapB, ACD,
310048. After ISH, cells were rinsed twice with PBS and blocked with Protein-
Block reagent (Dako, X090930-2) for 10 min, and IF was carried out with an Ecad
antibody (BD) diluted in antibody diluent (Dako, S302281-2) for 30 min; Ecad was
the junctional marker used here because it resisted protease treatment and did not
produce background due to formalin fixation. Cells were then washed three times
with PBS and stained with a fluorescent-labelled secondary antibody and DAPI for
30 min. Cells were finally washed three times with PBS, mounted (Aqua Poly/Mount,
Polysciences) and imaged using a Leica DM5000B microscope under bright (ISH)
and fluorescent light (protein IF). All images were pseudocoloured and merged
images were created using the microscope’s Leica suite software.
NanoString miRNA analysis. Total RNA was isolated as above and global miRNA
analysis was carried out using the nCounter Human v2 miRNA expression assay
(NanoString) containing 735 miRNAs. Data were collected using the nCounter
Digital Analyzer (NanoString). miRNAs were first elongated by a sequence-specific
ligation and were then hybridized with specific, barcoded probes. The raw units
that result from the NanoString reaction reflect the number of times each probe is
counted in a sample of the total hybridization reaction and represent the frequency
of each miRNA, without amplification of starting material. The raw units were then
normalized for RNA input and background noise to internal positive and negative
controls according to the manufacturer’s instructions. miRNAs with low counts at
the level of the negative controls were excluded.
Northern blot. 10–20 µg of total RNA or the Low Range ssRNA Ladder (NEB)
were mixed in a 1:1 volume ratio with Gel Loading Buffer II (Life Technologies),
denatured at 95 ◦
C for 5 min and loaded onto 12% denaturing PAGE RNA
gels (SequaGel; National Diagnostics). RNAs were then transferred to Hybond
N+ membranes (Amersham, GE Healthcare) for 2 h at 250 mA using a semi-
dry apparatus (Hoefer TE70) and cross linked using a Stratalinker (Stratagene).
Northern blot was carried out using the DIG Northern Starter Kit (Roche) and the
DIG Wash and Block Buffer Set (Roche) according to the manufacturer’s protocol.
Hybridization and stringent washes were carried out at 50 ◦
C; probes were used
at 5 nM final concentration. Probes used: hsa-miR-30b miRCURY LNA Detection
probe, DIG labelled, Exiqon, 18143-15; U6 hsa/mmu/rno control, miRCURY LNA
detection probe, DIG Labelled, Exiqon, 99002-01.
Statistics and reproducibility. For all quantitative experiments, averages and s.d.
were calculated and presented as error bars, whereas the number of independent
experiments carried out and the related statistics are indicated in each figure
legend. Student’s two-tailed t-test was employed for P-value calculations because
all comparisons were between two groups, control and experimental condition. For
all other experiments, at least three independent experiments were carried out and a
representative is shown in Figs 1a–h, 2b,d–f, 3b (left panel), 3d–g, 4b–g, 5c,d, 6b–k,
7a–i and 8a,c,d,f; and Supplementary Figs 1a–j, 2a–c, 3b–i, 4c–e, 5b–d,g–i, 6a–h and
7a–d,f–h,j–m.
Accession numbers. The NanoString data generated for this manuscript were
submitted to the Gene Expression Omnibus database, accession number GSE61593.
57. Smith, A. L., Friedman, D. B., Yu, H., Carnahan, R. H. & Reynolds, A. B. ReCLIP
(reversible cross-link immuno-precipitation): an efficient method for interrogation of
labile protein complexes. PLoS ONE 6, e16206 (2011).
58. Smoot, M. E., Ono, K., Ruscheinski, J., Wang, P. L. & Ideker, T. Cytoscape 2.8:
new features for data integration and network visualization. Bioinformatics 27,
431–432 (2011).
NATURE CELL BIOLOGY

S UPP LE ME NTARY INFORMATION
WWW.NATURE.COM/NATURECELLBIOLOGY 1
DOI: 10.1038/ncb3227
EcadPLEKHA7
Caco2
p120PLEKHA7
MDCK
a b
d PLEKHA7
x
z
Myosin
IIA
c
Actin
x
z
Afadin
apicalbasal
apicalbasal
y
PLEKHA7 p120:Y96
e
x
x
y
x
y
x
z
EcadPLEKHA7
MDCK
x
z
apicalbasal
apicalbasalapicalbasal
f
Caco2
p120:Y228PLEKHA7
MDCK
x
y
x
y
x
z
x
y
p120:T310PLEKHA7 merged
x
y
x
z
Rac1PLEKHA7
ih
PLEKHA7
MDCKCaco2
RhoAPLEKHA7
Caco2
j
Rac1
apicalbasal
apicalbasal
apicalbasal
x
y
x
y
x
y
g
merged
Caco2
Caco2
Supplementary Figure 1
Supplementary Figure 1 Distinct complexes exist at the junctions of
epithelial cells. Caco2 or PLEKHA7-GFP transfected MDCK cells were
grown to polarize, subjected to IF for PLEKHA7 or GFP respectively
and co-stained for (a) p120; (b,c) E-cadherin (Ecad); (d) Afadin,
Actin (phalloidin), Myosin IIA; (e) phosphorylated p120: Y228; (f)
phosphorylated p120: Y96; (g) phosphorylated p120: T310; (h,i) Rac1; (j)
RhoA. Stained cells were imaged by confocal microscopy and image stacks
were acquired, as in Figure 1. Representative x-y image stacks are shown
and/or the merged composite x-z images. Scale bars for x-y images: 20 μM;
for x-z images: 5 μM.

SUPPLEMENTARY INFORMATION
2 WWW.NATURE.COM/NATURECELLBIOLOGY
NT
PLEKHA7Ecad merged
shEcad
NT
PLEKHA7p120 merged
sh120+1A
shp120
sh120+4A
c
b
WBs
α-catenin
IPs
Ecad
β-catenin
a
Supplementary Figure 2 PLEKHA7 localization to the junctions is
E-cadherin- and p120-dependent. (a) Western blot of the lysates from the
separated apical and basolateral fractions shown in Figure 2 for E-cadherin
(Ecad), α-catenin, and β-catenin. (b) Caco2 control (NT) or E-cadherin
knockdown (shEcad) cells, stained by IF for E-cadherin and PLEKHA7.
(c) p120 and PLEKHA7 IF stainings of Caco2 control (NT) cells, p120
knockdown (shp120) cells, and of p120 knockdown cells transfected either
with the full length murine mp120-1A isoform (shp120+1A) or the murine
mp120-4A isoform that lacks the N-terminal PLEKHA7-binding domain
(shp120+4A). All scale bars: 20 μM.

0
0.2
0.4
0.6
0.8
1
1.2
NT #8 #10
foldchange
shPLEKHA7
PLEKHA7
mRNA
* *
a c
d
#8 #10NT
shPLEKHA7
PLEKHA7
p120
NT shPLEKHA7
merged
y
z
x
b soft agar colonies
PLEKHA7
p120
p-p120:
Y228
Src
p-Src:
Y416
SNAI1
Actin
MDCK
NEZHA
p120
p-p120:
Y228
Src
p-Src:
Y416
SNAI1
MYC
CCND1
Cad11
NT
shNEZHA
#72 #73
g
PLEKHA7
p120
Src
SNAI1
MYC
CCND1
p-Src:Y416
h
mp120 PLEKHA7
ΔARM1
1A
f
NT
shNEZHA
NEZHAPLEKHA7
i
adGFP
adSNAI1
soft agar
colonies
e
Actin
Actin
* **
Supplementary Figure 3 PLEKHA7 loss from the junctions results in
increased anchorage-independent growth and related signalling. (a)
Demonstration of the PLEKHA7 mRNA knockdown by qRT-PCR after
infection of Caco2 cells with two PLEKHA7 shRNAs (shPLEKHA7 #8 and
#10) or non-target control shRNA (NT) (mean ± s.d. from n=3 independent
experiments; *P0.0001, Student’s two-tailed t-test). Source data are
provided in Supplementary Table 2. (b) Caco2 control (NT) and PLEKHA7
shRNA knockdown (shPLEKHA7) cells were stained and imaged for
PLEKHA7 and p120. (c) Caco2 control (NT) or PLEKHA7 knockdown cells
(shPLEKHA7 #8, #10) grown on soft agar and imaged for colony formation
(images are in 2x magnification; see Fig. 3c for quantitation). (d) MDCK cells
were infected with either control (NT) or PLEKHA7 shRNA (shPLEKHA7#8)
and subjected to western blot for the markers shown. Phosphorylation
sites are denoted by p-. Actin is the loading control. (e) Soft agar assay
of Caco2 cells infected with either vector control (adGFP) or a SNAI1-
expressing construct (adSNAI1) (images are in 2x magnification; see Fig.
3h for quantitation). (f) Western blot of control (NT) or NEZHA knockdown
(shNEZHA#72 and #73 shRNAs) Caco2 cells for the markers shown; Actin is
the loading control. (g) IF of control (NT) or NEZHA knockdown (shNEZHA)
Caco2 cells for PLEKHA7 and NEZHA. (h) IF of Caco2 cells transfected
with either wild type murine mp120-1A (1A) or the murine mp120-ΔARM1
(ΔARM1) construct that cannot bind E-cadherin, stained with the murine-
specific p120 antibody 8D11 (mp120) and co-stained with PLEKHA7.
(i) Western blot of pcDNA (vector control), mp120-ΔARM1, or mp120-1A
transfected cells, for the markers shown; Actin is the loading control. Single
and double stars on the p120 blot indicate the 1A and ΔARM1 bands,
respectively, right above the endogenous p120 bands. Scale bars for x-y
images: 20 μM; for x-z images: 2.5 μM; for panels c and e: 2 mm.

0%
20%
40%
60%
80%
100%
PLEKHA7 p120 Ecad
%cases
absent mislocalized normal
DCISnormal
stage 2normal
BC
RCC
a
b
p120EcadPLEKHA7 Triple neg.
stage 3
0%
20%
40%
60%
80%
100%
PLEKHA7 p120 Ecad
%cases
absent mislocalized normal
p120EcadPLEKHA7
c
shp120
shPLEKHA7
-- +
- + +
soft agar colonies
d
shp120
shPLEKHA7 - -
--
+
+
+
+
α-Tubulin
PLEKHA7
p120
Ecad
p120:Y228p120
PP2
control
e
Supplementary Figure 4 PLEKHA7 is mis-localized or lost in breast and
renal tumour tissues. Representative immunohistochemistry images of
(a) breast and (b) kidney (renal), normal and cancer tissues stained for
PLEKHA7, p120 and E-cadherin (Ecad) (left panels) and the percentage
of tissues that exhibit presence, absence, or mis-localization of the three
markers examined (right panels). BC: breast cancer; RCC: renal cell
carcinoma. Scale bars: 20 μM. Number of tissues examined per cancer
type/stage; BC TMA: Benign, n=8; DCIS, n=12; ILC ER+ Her-, n=10; IDC
ER+, Her-, n=16; IDC Her+, n=16; IDC Triple Negative, n=13; RCC TMA:
Matched normal, n=119; stage 1, n=71; stage 2, n=20; stage 3, n=22;
stage 4, n=6. (c) Caco2 control (NT), PLEKHA7 knockdown (shPLEKHA7),
and PLEKHA7, p120 (shPLEKHA7, shp120) double knockdown cells were
grown on soft agar for colony formation assay (images are shown in 2x
magnification; see Fig. 4a for quantitation) and (d) examined by western
blot for E-cadherin (Ecad) levels; α-Tubulin is the loading control. (e) Caco2
cells treated with either vehicle (DMSO) or the Src inhibitor PP2 (10 μM)
were stained for p120 and phosphorylated p120: Y228. Scale bars 20 μM;
for panel c: 2 mm.

0
0.5
1
1.5
2
2.5
3
3.5
4
NT shPLEKHA7 shPLEKHA7+LZRS-PLEKHA7
foldchange
miR-19a
0
0.2
0.4
0.6
0.8
1
1.2
NT shPLEKHA7 shPLEKHA7+LZRS-PLEKHA7
foldchange
miR-30b
0
0.5
1
1.5
2
2.5
SNAI1 CCND1 c-Myc
foldchange
mRNAs
NT
shPLEKHA7
PLEKHA7
SNAI1
MYC
CCND1
Actin
0 15 30 60 240120
NT shPLEKHA7
CHX (min):
MG-132 (min):
PLEKHA7
SNAI1
MYC
CCND1
Actin
PLEKHA7
GSK3β
Actin
p-GSK3β
NT
shPLEKHA7
#8#10
dc
a
g
0 15 30 60 240120
0 15 30 60 240120
NT shPLEKHA7
0 15 30 60 240120
b
Src
p120
Src
p120
p-p120:
Y228
p-Src:
Y416
p-p120:
Y228
p-Src:
Y416
anti-miRs miR-mimics
h
NT shPLEKHA7
LZRS-PLEKHA7
-
--
+
+
+
*
LZRS-PLEKHA7
shPLEKHA7 -
--
+
+
+
*f
e
p120
p-p120:
Y228
i
Actin
shPLEKHA7
Supplementary Figure 5Supplementary Figure 5 PLEKHA7 acts via miRNAs but not post-
translational modification mechanisms. (a) qRT-PCR analysis for the
indicated mRNAs of Caco2 control (NT) or PLEKHA7 knockdown cells
(shPLEKHA7) (mean ± s.d. from n=3 independent experiments). (b)
Western blot for the markers shown (Actin: loading control) of control (NT)
or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells treated with 20 μg/ml
cycloheximide (CHX) or (c) 10 μM MG-132, for the indicated time points.
(d) Western blot of control (NT) or PLEKHA7 knockdown (shPLEKHA7)
Caco2 cells for the markers shown; Actin is the loading control. (e,f) miR-
30b and miR-19a qRT-PCR analysis of PLEKHA7-knockdown (shPLEKHA7)
Caco2 cells after ectopic re-expression of PLEKHA7 (LZRS-PLEKHA7)
(mean ± s.d. from n=3 independent experiments; *P0.05, Student’s
two-tailed t-test) (g) Caco2 cells transfected with the indicated anti-miRs
(a-miR) were subjected to western blot for the markers shown. (h) Caco2
control (NT) and PLEKHA7 knockdown (shPLEKHA7) cells were transfected
with either control or miR-30b mimic and blotted for the markers shown.
(i) Caco2 cells infected with either vector control (adGFP) or a SNAI1-
expressing construct (adSNAI1) were subjected to western blot for the
markers shown; Actin is the loading control. Source data for panels a, e, f
are provided in Supplementary Table 2.

mergedDROSHA*
apicalbasal
Caco2
*2nd ab
c
e
apicalbasal
mergedDGCR8*p120
*2nd ab
a
d
DROSHA
siDROSHA
NT
DGCR8
siDGCR8
NT
DROSHA
Actin
DGCR8
Actin
113
47
kDa
202
73
kDa
113
73
37
37
f g
Caco2
202
21
50
80
nt:
U6
pre-
miR-30b
mature
miR-30b
b
p120
p120 p120
mergedDROSHA
MDCK
apicalbasal
p120 mergedDGCR8
MDCK
apicalbasal
p120
h
PLEKHA7
p120
WBs
Ecad
IPs
Supplementary Figure 6 The microprocessor complex localizes at the
ZA. (a) Northern blot analysis of control (NT) or PLEKHA7 knockdown
(shPLEKHA7) Caco2 cells using a miR-30b probe, indicating the pre-
miR-30b and the mature miR-30b. U6 is the loading control. (b,c) IF of
polarized Caco2 cells for p120 and DROSHA or DGCR8. Antibodies used
here: DROSHA: Sigma and DGCR8: Sigma. The indication *2nd ab refers to
the use here of a different antibody for DROSHA and DGCR8, compared to
the antibodies used in Fig. 6d,e (for antibody details, see Supplementary
Table 3). Enlarged details in boxes are shown on top of apical fields. The
x-z composite image of panel b is shown in Fig. 6f and of c in Fig. 6g. (d,e)
IF of polarized MDCK cells for p120 and DROSHA (antibody: Abcam) or
DGCR8 (antibody: Abcam). (f,g) siRNA-mediated knockdown of DROSHA
(siDROSHA) and DGCR8 (siDGCR8) in Caco2 cells, shown both by IF (left
side of each panel), and western blot (right side of each panel). Non-target
(NT) siRNA is the control. p120 was used as a co-stain for the IF; Actin is
the loading control for the blots; molecular weights (kDa) are indicated on
the left side of each blot. (h) Western blots of PLEKHA7, p120, DROSHA,
and DGCR8 IPs for PLEKHA7, p120 and E-cadherin (Ecad). IgG is the
negative IP control. Scale bars: 20 μM; for enlarged parts of panels b and
c: 3 μM.

0.0
0.2
0.4
0.6
0.8
1.0
1.2
foldchange
NT shp120
NT
shPLEKHA7
shPLEKHA7 + shp120
DROSHA DGCR8p120
NT
shPLEKHA7
shPLEKHA7 + shp120
NT
shPLEKHA7
shPLEKHA7 + shCad11
Cad11
DROSHA DGCR8PLEKHA7
control
Nocodazole
NT
shNEZHA
NEZHA DROSHA NEZHA DGCR8
NT
shNEZHA
g h
fd
j k l m
NT
shPLEKHA7
shPLEKHA7 + shCad11
DROSHA
NT
shp120
i
e
DROSHA DGCR8Cad11p120
p120
* * * *
mp120 DROSHA
ΔARM1
1A
mp120 DGCR8
ΔARM1
1A
a b cmp120 PLEKHA7
ΔARM1
1A
Supplementary Figure 7 Regulation of the microprocessor at the ZA is
PLEKHA7-depended. (a-c) IF of Caco2 cells transfected with either the wild
type murine mp120-1A (1A) construct or the murine mp120-ΔARM1 (ΔARM1)
construct that cannot bind E-cadherin, both stained with the murine-specific
p120 antibody 8D11 (mp120) and co-stained with PLEKHA7, DROSHA, or
DGCR8. Arrows indicate affected junctional staining of the indicated markers
in transfected cells. (d) IF of control (NT) or p120 knockdown (shp120)
Caco2 cells for DROSHA and p120. (e) qRT-PCR of control (NT) and p120
knockdown (shp120) Caco2 cells for the miRNAs shown (mean ± s.d. from
n=3 independent experiments; *P0.05, Student’s two-tailed t-test). (f) IF of
control (DMSO) or Nocodazole (10 μΜ for 8h) treated Caco2 cells for PLEKHA7,
DROSHA, or DGCR8. (g,h) IF of control (NT) or NEZHA knockdown (shNEZHA)
Caco2 cells for DROSHA and DGCR8, co-stained with NEZHA. (i) qRT-PCR
of control (NT), PLEKHA7 knockdown (shPLEKHA7), PLEKHA7+p120
(shPLEKHA7+shp120), and PLEKHA7+Cadherin-11 (shPLEKHA7+shCad11)
double knockdown Caco2 cells for the indicated miRNAs (individual data
points and mean from n=2 independent experiments are shown). (j,k) IF of
control (NT), PLEKHA7 knockdown (shPLEKHA7), and PLEKHA7+p120
double knockdown (shPLEKHA7+shp120) Caco2 cells for DROSHA
and DGCR8, co-stained with p120. (l,m) IF of control (NT), PLEKHA7
knockdown (shPLEKHA7), and PLEKHA7+Cadherin-11 double knockdown
(shPLEKHA7+Cad11) Caco2 cells for DROSHA and DGCR8, co-stained with
Cadherin 11 (Cad11). Non-specific cytoplasmic or nuclear background appears
respectively for the two Cadherin-11 antibodies used in the IF (see Methods
for antibody details). All scale bars: 20 μM. Source data for panels e and i are
provided in Supplementary Table 2.

114
73
118
Fig. 3d
206
97
kDa
37
PLEKHA7
Actin
p120
Actin
p-p120:Y228
54
97
118
206
97
37
54
29
118
206
97
54
118
206
97
#8NT
shPLEKHA7
#10
#8NT
shPLEKHA7
#10
p-p120:Y96
p-
p120:Y228
114
202
73
114
202
73
p120
114
202
CCAR1
73
114
202
ACTN1
202 IQGAP1
73
114
202
PLEKHA7
73
114
EWS
28
36 HNRNP
A2B1
53
28
36
TRIM2153
IPs
Fig. 2dkDa Fig. 3e
p130CAS
Src
#8NT
shPLEKHA7
#10
118
206
97
kDa
37
54
97
37
54
118
206
97
37
54
29
17
p-p130CAS:
Y165
p-Src:
Y416
Actin
77
Fig. 3f
203
117
77
SNAI1
Cad11
36
52
28
36
52
28
36
52
28
36
52
28
CCND1
MYC (~62kDa)
77
52 VIM
203
117 FAK
Actin
#8NT
shPLEKHA7
#10
kDa
PLEKHA7
203
117
PLEKHA7118
206
97
- +
Fig. 3g
33
47
27
33
47
27
PLEKHA7
Actin
p120
Cad11
MYC
SNAI1 CCND1
47
72
72
202
114
72
202
114
72
202
114
72
202
114
33
47
27
LZRS-PLEKHA7-myc
shPLEKHA7 -
-
+ +
kDa
p-p120:
Y228
72
202
114 p-p120:
Y96
Fig. 3h
36
53
78
28
α-Tubulin
SNAI1
short
exposure
77
Supplementary Figure 8 Full western blot scans. Multiple experiments
were routinely run on every gel and the membranes were cut and blotted for
multiple antibodies for each experiment. The cut blots for each antibody
are shown here per case. Molecular weights are indicated on the left; the
cropped images shown in the respected Figure panels are indicated in yellow
boxes.

Fig. 4b Fig. 4d
PLEKHA7
p120
SNAI1
CCND1
Cad11
MYC
Actin
79
205
113
78
211
118
78
211
118
36
53
36
28
36
28
53
78
shp120
shPLEKHA7-
-- +
+ +
72
202
114
PLEKHA7
p120
72
202
114
72
202
114
33
27
SNAI1
33
27
CCND1
33
27
Actin
47
72
202
114
Cad11
MYC
33
27
47
72
PP2
shPLEKHA7 - -
-
+
+
+
-+
kDa kDa
p-p120:Y228
(part 2)
Fig. 4c
shEcad
shPLEKHA7-
-- +
+ +
PLEKHA7
Ecad
SNAI1
CCND1
Actin
79
205
113
17
34
27
17
34
27
37
79
47
MYC
(~62kDa)
37
47
p-p130CAS:
Y165
Fig. 4e
78
211
118
kDa
p120
Actin
p120
SNAI1
Cad11
78
211
118
28
53
36
78
211
118
78
211
118
53
36
p-p120:
Y228
-
17
Fig. 4f
Actin
SNAI1
Cad11PLEKHA7
78
211
118
MYC
CCND1
28
53
36
28
53
36
36
53
78
shCad11
shPLEKHA7 - -
-
+
+
+
-+
- -
-
+
+
+
+
kDa
p130CAS
p-p130CAS:
Y165
72
202
114
72
114
202
p130CAS
78
211
118
78
211
118
78
211
118
Fig. 5c
SNAI1
CCND1
Actin
50
75
25
37
anti-
miRs:
kDa
50
MYC
(~62kDa)
25
37
50
p130CAS
p-p130CAS: Y165
205
113
79
205
113
79
Supplementary Figure 8 continued

77
114
non-
nuclear
Fig. 5d
Actin
SNAI1
CCND1
MYC
34
47
73
27
34
27
kDa
27
47
34
miR-mimics
NT shPLEKHA7
Fig. 6b
6
47
17
73
202
114
DROSHA
DGCR8
Lamin A/C
Histone H3
GAPDH
Actin
73
114
kDa
27
47
33
47
33
nucleartotal
Fig. 6h
p120
DROSHA
DGCR8
PLEKHA7
202
114
77
114
202
114
IPs
Fig. 6i
kDa
77
114
p120
PLEKHA7202
114
77
114
202
114
kDa
202
Fig. 6j
202
114
73
202
114
114
DROSHA
DGCR8
PLEKHA7
IPs
Fig. 8a
kDa
kDa
DROSHA
DGCR8
Lamin A/C
Histone H3
GAPDH
202
114
73
DGCR8
202
114
73
6
47
17
73
27
47
33
short exposure:
PLEKHA7
47
114
74
201
Supplementary Figure 8 (part 3)
202
77
IPs
DROSHA
DGCR8
DROSHA
Supplementary Figure 8 continued

Supplementary Table Legends
Supplementary Table 1 The proteins identified by proteomics of junctional fractions.
Supplementary Table 2 The source data file.
Supplementary Table 3 The antibodies used in the study.

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