The document describes the Cignal 10-Pathway Reporter Array, a tool that can simultaneously measure the activity of 10 cancer-related signaling pathways in a single experiment. The array contains reporter assays to analyze pathways such as Wnt, Notch, p53, TGFß, NFkB, and MAPK. Results showed that knockdown of the tumor suppressor p53 downregulated p53 signaling but upregulated Notch, hypoxia and MAPK/ERK pathways in HEK293 cells. As Notch signaling is often deregulated in cancer, this suggests Notch may act as an oncogene. Knockdown of Dicer, required for siRNA and miRNA pathways, downregulated Notch,

1. Cancer 10-Pathway Reporter Array: An innovative tool to simultaneously
measure the activities of ten signaling pathways involved in cancer biology
Abigail Harris and Vikram Devgan
Abstract
10-Pathway Reporter Array
The Cignal 10-Pathway Reporter Array consists of 10 dual-luciferase reporter assays along with negative
and positive controls. This product enables a complete analysis of the activity of multiple pathways within
a single experiment. Arrays are delivered as a 96-well tissue culture plate, with reporter or control
constructs dried down in each column of the plate (see Cancer array content and layout below).
1
2
3
4
5
6
7
8
9 10 -ve +ve
Transcription factor
A.
TATA
box
Luciferase or GFP
Tandem repeats of TREs
B.
CMV immediate early
enhancer/promoter
Renilla
Luciferase or GFP
D.
CMV immediate early
enhancer/promoter
Luciferase
E.
CMV immediate early
enhancer/promoter
MGFP
Cancer 10-Pathway Reporter Arrays were used to understand the effects of p53 (I) and Dicer (II) knockdown on
ten cancer signaling pathways. HEK-293H cells were reverse transfected with p53 siRNA or negative control siRNA
(I), and Dicer siRNA or negative control siRNA (II) using the 10-pathway reporter array. Forty-eight hours after
transfection, dual-luciferase assay (Promega) was performed. Promoter activity values are normalized using a Renilla
reporter activity and expressed as fold change compared to negative control siRNA.
I. p53 siRNA
Performance Data
II. Dicer siRNA
HEK-293H cells were reverse transfected using the Cignal Cancer 10-Pathway Reporter Assay. After 16 hours of transfection, medium was
changed to complete medium (DMEM containing 10% of fetal bovine serum, 1% NEAA, 100 U/ml Penicillin and 100 µg/ml Streptomycin).
Twenty-four hours after transfection, cells were treated with different stimuli as described above. Forty-two hours after transfection, dualluciferase assay (Promega) was performed. Promoter activity values are normalized using a Renilla reporter activity and expressed as fold change
compared to non-stimulated control.
The reporter assay showed high induction in the activity of the Cignal reporters in response to a specific stimulus for their corresponding
signal transduction pathways. Non-responsiveness of HIF reporter is a 293H cell-specific effect.
The reporter assay reported 3-fold decrease in p53 signaling by p53siRNA. Moreover, p53siRNA induced Notch,
hypoxia and MAPK (ERK) signaling. This is consistent with previous reports that p53 inhibits Notch, hypoxia and
ERK signaling. Likewise, Dicer-specific siRNA showed down-regulation of the Notch, p53, NFkB, Myc/Max and
MAPK/JNK pathways. This data indicates the effectiveness of the Cignal 10-Pathway Reporter Array for sensitive,
specific and rapid screening siRNA effects even in a basal (non-stimulated) condition.
The inducible transcription factor responsive construct
(TREs-luc/GFP)
Conclusions
The constitutively expressing Renilla construct (CMV-Renilla)
TATA
box
C.
How It Works:
Column Pathway (Transcription Factor)
1. Wnt (TCF/LEF)
2. Notch (RBP-jk)
3. p53/DNA damage (p53)
4. TGF (SMAD2/3/4)
5. Cell cycle/pRb-E2F (E2F)
6. NF B (NF B)
7. Myc (Myc)
8. Hypoxia (HIF1A)
9. MAPK/ERK (Elk-1/SRF)
10. MAPK/JNK (AP1)
11. Cignal Negative Control
12. Cignal Positive Control
κ
Pathway Reporter Assay: Technology Overview
The CignalTM Reporters are transcription factor reporter assays that measure effects of signal
transduction in cultured cells. Signaling pathways regulate gene expression via associated
transcription factors which bind specific transcription response elements (TREs). Detection of
transcription factor activity is a reliable way to monitor the intracellular status of many signaling
pathways. Transcription factor reporter assays place reporter genes, such as luciferase or GFP,
under the control of transcription factor-specific TREs. Therefore, alteration in reporter gene
activity signals change in the activity of transcription factors and their associated pathways. The
Cignal Reporters are engineered for accurate, sensitive and quantitative measurement of
transcription factor activity, and can be used to determine the effects of siRNAs, small chemicals
and full length cDNA clones on multiple signaling pathways in a variety of cells.
Schematic representation of Pathway Reporter Assay constructs:
Results
Content of Cancer 10-Pathway Reporter Array:
.
TRE: Multiple repeats of pathway-specific transcription factor
regulatory element.
Reporter : Luciferase or GFP
Frederick, MD 21703 USA
β
Most cancers are the result of de-regulated cell signaling pathways. Typically, more than one signal
transduction pathway is involved in cancer pathogenesis and progression. In order to fully
understand the physiology of cancer, it is vital to understand the intracellular status of multiple
signaling pathways. We have developed a Cancer 10-Pathway Reporter Array to simultaneously
measure the activities of ten key cell signaling pathways, including Notch, Wnt/β-Catenin, TGFβ,
p53, NFkB, E2F, MAPK (JNK), MAPK (ERK), Myc and HIF-1α, which serve as critical regulators
of a cancer biology. In this study, we utilized the cancer 10-pathway reporter array to determine the
effect of the knock down of well-known tumor suppressor gene p53 on important cancer signaling
pathways. Our results showed that the knock down of p53 gene expression down-regulates p53
signaling, while up-regulating Notch, hypoxia and MAPK/ERK signaling in HEK-293 cells.
Interestingly, Notch signaling is known to be frequently deregulated in human malignancies. Upregulation of Notch signaling by p53 RNA interference suggests that Notch may function as a protooncogene in HEK-293 cells. Moreover, our data potentiate the ability of the reporter array to rapidly,
accurately and reproducibly quantify the impact of a given stimulus (RNAi/small chemical
molecules/antibodies) on the activities of ten cancer signaling pathways in a single experiment.
SABiosciences 6951 Executive Way
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Abstract #: 5253
The non-inducible reporter construct (Pmin-luc/GFP)
The constitutively expressing luciferase construct (CMV-luc)
The constitutively expressing GFP construct (CMV-GFP)
The Cignal 10-Pathway Reporter Array is an innovative tool for measuring the activity of 10 different signaling pathways in a single experiment.
Use of the Cancer 10-Pathway Reporter Array (luc) revealed that knockdown of p53 gene expression down-regulates p53 signaling, while Notch, hypoxia and MAPK/ERK signaling are up-regulated in HEK293H cells. As Notch signaling is known to be deregulated in many human cancers, up-regulation of Notch signaling by p53 RNA interference suggests that Notch may act as a proto-oncogene.
Dicer is known to be required in both siRNA and miRNA pathways, down regulation of Notch, p53, Myc and MAPK/JNK pathways by Dicer-specific siRNA suggests that these signaling pathways are heavily modulated by
microRNA and/or siRNA processing.
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