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a.
b.
Relative
mRNA
Levels
8
Relative 5
mRNA
Levels 4
6
3
Stat 1
4
Stat 1
2
WT1
2
1
0
0
M
A
B
M
10
25
50
75
nM TLR3 siRNA
Treatment
5nM WT1 siRNAs
Treatment
c.
d.
nM HPV-E7 siRNA
M 1 10
25 50 75 100 C
C
1
nM PKR siRNA
10
25
50 75
100
Stat 1
Stat 1
GAPDH
GAPDH
Supplemental Figure1. Stat 1 upregulation in response to 5 unique siRNAs. All cells
were transfected according to the protocol described in the manuscript and analyzed 48 hours
post transfection. (a) Semi-quantitative RT-PCR analysis of WIT49 cells transfected with
5nM of 2 unique siRNAs, designated A and B, targeting WT1. (b) Semi-quantitative
RT-PCR analysis of T98G cells transfected with the indicated concentrations of a TLR3specific siRNA. The siRNA directed against TLR3 did not suppress its expression, but
did cause Stat 1 upregulation. (c) Western blot analysis of T98G cells transfected with the
indicated concentrations of anHPV E7 siRNA. This siRNA has no cellular target. (d) Western
Blot analysis of T98G cells transfected with the indicated concentrations of an siRNA targeting
PKR. (The siRNAs specific for TLR3, HPV-E7, and PKR were chemically synthesized and gel
Purified by Xeragon, Inc. The WT1 siRNAs were enzymatically synthesized with the Ambion
siRNA Silencer kit, according to the manufacturer’s protocol.)
a
ss C M 10 25 50 100 ss C M 10
ss C M
10
ss C
M 10
OAS2
ISG 15
RCC1
b
Relative
mRNA
Levels
T98G
RCC1
T98G
100
75
ISG 15
50
ISG 56-K
25
IFIT 1
0
C
M 10ss 10 25 50 100
nM GAPDH siRNA
RCC1 Cell Treatment
Relative
mRNA
Levels
125
100
ISG 15
75
ISG 56-K
50
IFIT 1
25
0
C
M
ss10
10
nM GAPDH siRNA
T98G Cell Treatment
Supplemental Figure 2. Verification of ISG Microarray Analysis. (a) Northern blot analysis
showing upregulation of two representative ISGs, OAS 2 and ISG 15, in response to
GAPDH siRNA in the two cell lines, RCC1 and T98G. (The numbers indicate the concentration
of siRNA; ss, 10nM single GAPDH sense strand 21-mer)
(b) Expression of three representative ISGs (ISG 15, ISG 56-K, and IFIT 1) from the ISG
microarray experiment were analyzed by semi-quantitative RT-PCR to confirm their upregulation
in response to siRNA transfection. The top panel shows the response seen in RCC1 cells.
The bottom panel shows the response seen in T98G cells.
nM GAPDH
siRNA
M
ss25
25
P-PKR (Thr 446/451)
Total PKR
Supplemental Figure 3. In vivo phosphorylation of PKR on threonine residues 446/451
in response to GAPDH siRNA. T98G cells were transfected with the indicated concentrations
of either the single stranded 21-mer or the double stranded GAPDH siRNA at the
indicated concentration. Whole cell lysates were collected 90 minutes post transfection
and a PKR immunoprecipitation was performed accounding to the protocol described in the
manuscript. The siRNA caused PKR autophosphorylation on thr 446 and 451 to levels
above both single stranded and mock transfected.
a
Normalized
GL3 Luciferase
1.6
1.4
1.2
1
21bp GL3 siRNA
0.8
21bp RL siRNA
0.6
0.4
0.2
0
WT MEF
IRF9
Stat1
Null
Null
Cell Lines
Type I IFN
Null
b
0.3
Normalized
GL3 Luciferase
0.25
0.2
500bp IR RNA
0.15
500bp IR RNA
0.1
0.05
0
WT MEF
IRF9
Stat1
Null
Null
Cell Lines
Type I IFN
Null
Supplemental Figure 4. RNA interference of GL3 luciferase by 21bp siRNA in mutant cell lines.
Fold change of GL3 luciferase for each cell line was normalized to its own reporter control
(pGL3-Control + pRL-SV40), which was given a value of 1. The graphs represent data averaged from
at least 3 independent experiments +/- standard deviation. (a) 21 bp siRNA directed against GL3
luciferase mediated sequence specific silencing while (b) 500 bp IR RNAmediated non-specific
silencing of GL3 luciferase in both wild type cells and in cells lacking either IRF 9, Stat 1, or Type I
IFN.