0
50
100
150
200
250
300
0
500
1000
1500
2000
2500
Re
lati
ve
mR
NA
lev
el
0
200
400
600
800
1000
1200
0
400
800
1200
1600
2000
2400
0
5
10
15
20
25
30
35
40
IP10 ISG15 ISG54 OASL1 Mx1 Rantes
B
Untreated
200 U IFN
200 U IFN+Dex
500 U IFN
500 U IFN+Dex
1000U IFN
1000U IFN+Dex
A
IP10 ISG56 ISG15 ISG54 OASL1 Mx1 Rantes
0
5
10
15
20
25
30
C
0
2
4
6
8
IRF1
Untreated
IFNg
IFNg+Dex
Untreated
IFN 1h
IFN+Dex 1h
IFN 2h
IFN+Dex 2h
IFN 4h
IFN+Dex 4h
Re
lati
ve
mR
NA
lev
el
Re
lati
ve
mR
NA
lev
el
Supplementary Figure S1. Inhibition of ISGF3-mediated transcription by GCs is dose- and time-
dependent. BMM were treated with 500 U/mL IFN -/+ 100 nM Dex for indicated times (A) or
for 2 h with the indicated doses of IFN (B). (C) BMM were treated with IFN -/+ 100 nM Dex
for 2 h. mRNA expression of Type I IFN targets (A,B) or the IFN target, IRF1, (C) was assessed
by qPCR with GAPDH as a normalization control and expressed relative to untreated cells
(con=1).
F
F g
g
0
20
40
60
80
100
120
140
IP10 ISG56 Rantes ISG15 ISG54 IL6 OASL1 Mx10
200
400
600
800
1000
CXCL9 CXCL11
Re
lati
ve
mR
NA
le
ve
l
CHX
CHX+ IFN
CHX+ IFN+Dex
0
20
40
60
80
100
120
140
CXCL9 Rantes CXCL11 ISG15 ISG54 OASL1 Mx1
100
200
300
400
500
600
700
IP10rela
tiv
e n
as
ce
nt
tra
ns
cri
pt
lev
el
0
A
B
Untreated
IFN
IFN+Dex
Supplementary Figure S2. Dex-mediated suppression of ISG expression is independent of mRNA stability, processing, or new protein synthesis. (A) BMMF were treated with 500 U/mL IFN -/+ 100 nM Dex for 2 h, and nascent transcripts were analyzed by qPCR as in Figure 1, using primers specific for intronic regions. (B) BMMF were pretreated for 30 min with 20 mg/mL CHX, followed by treatment with 500 U/mL IFN -/+ 100 nM Dex, as indicated, and mRNA levels of target genes were analyzed by qPCR, as in Figure 1. Error bars represent ± SEM. Results are representative of at least three independent experiments.
0
1.5
3.0
4.5
6.0
IgG
STA
T2
IgG
STA
T2
IgG
STA
T2
IgG
STA
T2
Re
lati
ve
O
cc
up
an
cy
IP10 ISG15 ISG56 CXCL9
Untreated
IFN
IFN+Dex
0
5
10
15
20
25
30
35
40
45
IP10 ISG56
TSS
ISG15
ISRE
IP10
TSS
ISG56
ISRE
% In
pu
t
Pol2 GR
A
B
ISG150
5
10
15
20
25
30
35
40
GILZ GRE
GR
Untreated
IFN
IFN+Dex
Supplementary Figure S3. (A) IFN-induced STAT2 occupancy of ISREs is diminished by Dex co-treatment. BMMF were treated as indicated for 30 min. ChIPs were performed using antibodies to STAT2 or isotype-matched control IgG. Occupancy was determined by qPCR amplification over ISRE regions of indicated target genes and expressed as in Figure 3. Error bars represent ± SEM. Results are representative of three independent experiments. (B) GR is not recruited to ISG promoters. RAW264.7 cells were treated as indicated for 30 min. Occupancy of GR and Pol2, at the indicated regions of ISGs (left) and GILZ (right), was determined by ChIP, quantified by qPCR, and expressed as a percentage of total DNA input.
Untreated
Dex
- IFNIFN
Dex- IFN
IFN
Dex
WCE aGRIP1 IP
GRIP1
IRF9
Supplementary Figure S4. GRIP1 constitutively associates with IRF9 in mouse fibroblasts. 3T3 fibroblasts weretreated as shown for 1 h and lysates were prepared. 20% of each lysate was boiled in sample buffer to generate
WCE, whereas the rest was precipitated with anti-GRIP1 antibody ( GRIP1 IP). Protein complexes were collectedon protein A/G PLUS agarose beads, boiled in sample buffer and separated by SDS-PAGE along with WCE.GRIP1 and IRF9 were detected by immunoblotting.
a
Supplementary Table S1. qPCR primers used in the study
IP10-ChIP-ISRE F:5’-ATTCTGCAAGGCACTCATCTGATT-3’ R:5’-GTGACCCATGAACTTGGAATTTC-3’
IP10-ChIP-TSS F:5’-GGAGCCTTGCTGAGTCATCTCC-3’ R:5’-GGCAGCACTTGGGTTCATGGTGC-3’
IP10 exon F:5’-CTGCAACTGCATCCATATCG-3’ R:5’-CAATGATCTCAACACGTGGG-3’
IP10 intron F:5’-ACACAGACACTGAGGTGCCTTCTT-3’ R:5’-CATTTGGCAGCTTTACCCGTGACA-3’
ISG15-ChIP-ISRE F:5’-GGTTACAGCTTTGACCCTTGAGAGC-3’ R:5’-CGGAGTTTCCAGAAACCAGAGCTA-3’
ISG15-ChIP-TSS F:5’-CCGCCTCTTCACACCCACAGC-3’ R:5’-CTCTAGGTCCCCTGGAATTAAGGAG-3’
ISG15 exon F:5’-CAGGACGGTCTTACCCTTTCC-3’ R:5’-AGGCTCGCTGCAGTTCTGTAC-3’
ISG15 intron F: 5’- CTCTCCTCTATTAGGGGAACCATCCA-3’ R:5’-AGTTCAGGCCAAGTAGCTCGATGC-3’
ISG56-ChIP-ISRE F:5’-TCAGTGGAGAATGCAGTAGGGCAA-3’ R:5’-ACTGTCACACCAACTGGAAGCTCA-3’
ISG56-ChIP-TSS F:5’-TGAGCTTCCAGTTGGTGTGACAGT-3’ R:5’-CCTTACCCCATGGTTGCTGTAAAGG-3’
ISG56 exon F:5’-TGCTTTGCGAAGGCTCTGAAAGTG-3’ R:5’-TGGATTTAACCGGACAGCCTTCCT-3’
CXCL9-ChIP-ISRE F:5’-GTCATTGTATAATCTATTCCACATCCAGG-3’ R:5’-CTACTCTCAGATCCCAGGGAATTTC-3’
CXCL9-ChIP-TSS F:5’-TCAGCTGAGGAGACCAGCCAATC-3’ R:5’-GGACTTCATGGCAGAGCTGAGTTC-3’
CXCL9 exon F:5’-CCTTTCCTTCATAGCTATCCAATGCAC-3’ R:5’-CTCTCCAGCTTGGTGAGGTCTATC-3’
CXCL9 intron F:5’-CGTAGGTACCATCTGAGAGTGTAGG-3’ R:5’-CCCAGACAACTGACAAGTATGGC-3’
CXCL11-ChIP-ISRE F:5’-GTGGCTTCTTAACTCCAGCAGAGG-3’ R:5’-CCAGCAATCTTCAGGCAGTATCAGG-3’
CXCL11-ChIP-TSS F:5’-GAGAGATCTCCAAAGCCCAGGC-3’ R:5’-CAGACTCAGAAGCTACGGGCAC-3’
CXCL11 exon F:5’-ATGTGACATCCTGGGAACGTCTGAC-3’ R:5’-GTTCTGCAGCCTGGTAATACGTGG-3’
CXCL11 intron F:5’-TCACCAGCGACTGTGAAAGACGC-3’ R:5’-AGTATGAGTATGCTATGAGACCGGC-3’
ISG54 exon F:5’-GCCCACCCCTCCTCACAGTC-3’ R:5’-TATCACATGGGCCAGTTCTCAAAG-3’
ISG54 intron F:5’-AGTTCAGGCCAAGTAGCTCGATGC-3’ R:5’-GTTCTTCAACCTTGAACACTAACTCC-3’
OASL1 exon F:5’-GCCATTGCACGCTCGCCTACTAC-3’ R:5’-CTCCTGCCATCCGGGTTTTTCA-3’
OASL1 intron F:5’-AGGCTGAGAGTAGCCATTGGGCT-3’ R:5’-GAGAAACTTGGGTACCGTTTGAGTAG-3’
IL6 exon F:5’-ATGAAGTTCCTCTCTGCAAGAGACT-3’ R:5’-CACTAGGTTTGCCGAGTAGATCTC-3’
Mx1 exon F:5’-AAACCTGATCCGACTTCACTTCC-3’ R:5’-TGATCGTCTTCAAGGTTTCCTTGT-3’
Mx1 intron F:5’-CTGTCTACTGTAGGTGGAAGCATAGC-3’ R:5’-CTGCCTCATTTAAAGGTCAGGGTCC-3’
RANTES exon F:5’-GCCCCACGTCAAGGAGTATTTCTA-3’ R:5’-ACACACTTGGCGGTTCCTTC-3’
RANTES intron F:5’-CATATGTAGGAAAGCAGAGGGCAC-3’ R:5’-GACAATAGTACACGGTGTGGGTG-3’
GRIP1 F:5’-TGCTGCCAAAGTCCATGGTGAATG-3’ R:5’-ATCGCCTCGTATTTCTGATGGGCT-3’
Actin F:5’-AGGTGTGCACTTTTATTGGTCTCAA-3’ R:5’-TGTATGAAGGCTTTGGTCTCCCT-3’
GAPDH F:5’-ACGACCCCTTCATTGACC-3’ R:5’-AGACACCAGTAGACTCCACG-3’
28S-ChIP F:5’-GATCCTTCGATGTCGGCTCTTCCTATC-3’ R:5’-AGGGTAAAACTAACCTGTCTCACG-3’
GILZ-ChIP-GRE F: 5’-GGGACAGTGATTCACCCAACTCAG-3’ R: 5’-TTTCTCTTGGCCTGTTGGTCCTGC-3’