2. Testing the RV1B induced IRF3
activation
BEAS-2B
Infected with
-RV1B
-UV irradiated
RV1B
Collection of Cell
protein lysates after
12 hours
IRF3 or IFN-response factor 3 is a commonly expressed transcription factor for
regulating Type 1 IFN production.
3. Testing the RV1B induced IRF3
activation
IRF3 phosphorylation
was determined by
SDS-PAGE, followed
by immunoblotting
using anti-IRF3 Ab.
IRF3 dimerization
was determined by
native PAGE.
Poly (IC), a synthetic
dsRNA inducing both
IRF3 phosphorylation
and dimerization used
as positive control.
4. RV-induced IRF3 activation in cultured BEAS
Phosphorylated IRF3 protein at upper level and an
unphosphorylated IRF3 protein in the lower level
during immunoblotting.
Densitometry of IRF3 phosphorylation ratio using a Scanning
Electron Microscope (SEM) showing different levels in three
different experiments between responses against naïve, RV1B
and UV irradiated RV1B exposure.
Here the ratio between the phosphorylated and
unphosphorylated IRF3 is higher in case of RV1B than the
comparable low level for the UV RV1B infection.
5. RV-induced IRF3 activation in cultured BEAS
IRF3 dimerization from native-PAGE in two bands, upper one showing dimer and the
lower band showing monomer.
The naïve RV produces no IRF3 dimers.
It is notable that the dimers are absolutely absent in case of UV RV1B and the
reduction in RV1B while they are being compared with the positive control Poly (IC).
Although in these scenarios, monomers of IRF3 are also lesser than the naïve RV.
6. RV-induced IRF3 activation in cultured primary
airway epithelial cells (Tracheal epithelial cells)
After infecting primary tracheal epithelial cells with RV1B and UV RV1B , lysates
collection was done after 12 hours.
The upper band showing the lysates being
probed with anti-IRF3 Ab.
In the lower bands, cellular proteins were
subjected to native-PAGE to resolve the
dimerization of IRF3.
It actually shows a similar result as the experiments conducted using the BEAS-2B.
The requirement of IRF3 in RV-induced infection is thus proved by steering the same
experiment using two groups of cells.
7. For specific requirement of
IRF3 in RV-induced IFN
responses experiments
were done using siRNA
against IRF3.
BEAS-2B were infected
with either IRF3 siRNA or
nontargeting siRNA. Then
cells were infected with
RV1B, UV RV1B or sham.
8. siRNA against IRF3 blocks RV-induced IFN and
ISG expression
Cell lysates after infected by both IRF3 siRNA and
nontargeting siRNA were probed with anti-IRF3
Ab.
Then to see the effect immunoblotting was done.
IRF3 siRNA almost closed down the RV1B-induced
expressions of proteins.
9. siRNA against IRF3 blocks RV-induced IFN and
ISG expression (contd..)
After extraction of the total RNA from the lysates, the expression of several proteins were
determined by using quantitative PCR.
The expression of each target gene was normalized to Glyceraldehyde 3-phosphate
dehydrogenase or GAPDH.
In the bars, the y-axis has been broken from the basal and the maximum gene expression
to calculate each of their effects after infected with siRNA targeting each of them.
Fold increase for targeting siRNA expressions VS sham-infected, nontargeting siRNA-
transfected gene were recorded.
10. siRNA against IRF3 blocks RV-induced IFN and
ISG expression (contd..)
The expression for IFN-β shows that
the siRNA against it reduces it’s
expression.
Bars in the diagram represent Scanning Electron Microscope results of four experiments.
Number over bars represent fold increase of specific proteins compared with the sham-infected sample within its own siRNA
group.
11. siRNA against IRF3 blocks RV-induced IFN and
ISG expression (contd..)
The expressions of IFN-λ1, IFN-λ2/3, IRF-7, IP-10 were also demonstrated the same
nature after inducing specifically targeting siRNA during the experiment.
The IRF3 siRNA specifically against
IFN-λ1, IFN-λ2/3, IRF-7 and IP-10
markedly reduced their respective
effects.
Bars in the diagram represent Scanning Electron Microscope results of four experiments.
Number over bars represent fold increase of specific proteins compared with the sham-infected sample within its own siRNA
group.
12. siRNA against IRF3 blocks RV-induced IFN and
ISG expression (contd..)
But while using siRNA targeting IL-8 and GM-CSF, the experiment demonstrated no
effect of the siRNA for their expression.
Bars in the diagram represent Scanning Electron Microscope results of four experiments.
Number over bars represent fold increase of specific proteins compared with the sham-infected sample within its own siRNA
group.
13. From the diagrams we can conclude that,
• IRF3 siRNA reduced the protein abundance by IRF3.
• IRF3 siRNA almost abolished RV1B-induced expression of IFN-β, λ1, λ2/3,
IRF7 and CXCL 10
• IRF3 siRNA doesn’t affect the expression RV1B-induced expressions of IL-8
or GM-CSF.
14. Taken all the data into consideration
We can say-
• IRF3 are activated by RV1B
• For RV-induced IFN responses, IRF3 is necessary. For the response of ISGs
like IRF7 and IP-10, IRF3 is required as well.
