0

50

100

150

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300

0

500

1000

1500

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2500

Re

lati

ve

mR

NA

lev

el

0

200

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600

800

1000

1200

0

400

800

1200

1600

2000

2400

0

5

10

15

20

25

30

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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

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140

IP10 ISG56 Rantes ISG15 ISG54 IL6 OASL1 Mx10

200

400

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1000

CXCL9 CXCL11

Re

lati

ve

mR

NA

le

ve

l

CHX

CHX+ IFN

CHX+ IFN+Dex

0

20

40

60

80

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120

140

CXCL9 Rantes CXCL11 ISG15 ISG54 OASL1 Mx1

100

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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’