Fig 2: IFIT1 Modulates the Association of an LPS-Regulated HDAC2-SAP25 Protein Complex(A) THP1 cells stably expressing a SAP25-mCherry fusion protein were transfected with non-targeting control (NTC) or IFIT1 siRNA and stimulated with 100 ng/mL LPS for 0, 1, or 2 hr. The indicated proteins were detected by western blot in either a SAP25-mCherry immunoprecipitate (top) or in whole-cell lysates (bottom).(B) Cytosolic and nuclear fractions were isolated from 100 ng/ml LPS treated mCherry-SAP25 expressing cells and subjected to western blot analysis for Sin3A, HDAC2, mCherry, hnRNPL (nuclear marker) and GAPDH (cytosolic marker).(C and D) THP1 cells transfected with non-targeting control or HDAC2 siRNA were stimulated with 100 ng/mL LPS for the indicated times and mRNA levels of (C) TNF and (D) IFNB1 were measured by qPCR.Data are representative of three independent experiments. Data in (C) and (D) are expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-way ANOVA followed by Sidak’s multiple comparison test). See also Figure S4.

Fig 3: IFNB1 Induction Is Attenuated in IFIT1-Depleted Cells in Response to Multiple Different Stimuli(A–F) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with (A) 100 ng/mL LPS, (B) 10 µg/mL poly(I:C), (C) 10 µg/mL poly(dA:dT), (D) Sendai virus (Cantell) MOI 5, (E) influenza A virus (Texas/36/91) MOI 5, and (F) B. cenocepacia MOI 5, and IFNB1 mRNA was measured by qPCR. Data are representative of four (A) or three (B–F) independent experiments and are expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-way ANOVA followed by Sidak’s multiple comparison test).

Fig 4: Signaling Responses in IFIT1-Depleted Cells(A) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and cytosolic and nuclear fractions were subjected to western blot analysis for phos-pho-p65/NF-?B (Ser536), phospho p38 and MAPK (Thr180/Tyr182), p105/NF-?B, and p50/NF-?B. GAPDH and heterogenous nuclear ribonucleoprotein L (hnRNPL) were detected as positive controls for cytosolic and nuclear fractions.(B) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-IRF3 (S386), phospho-STAT1 (Tyr701), IFIT1, and actin (loading control).(C) WT and Ifit1-/- BMDM cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-STAT1 (Tyr701), phospho-p38 and MAPK (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK (Thr202/Tyr204), and GAPDH (loading control).(D) WT and Ifit1-/- mouse BMDM transfected with non-targeting control (NTC) or IFIT3 siRNA were stimulated with 100 ng/mL LPS and Ifnb1 mRNA was measured by qPCR.Data are representative of three independent experiments. Data in (D) are expressed as mean ± SD; **p < 0.01, ***p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparison test). See also Figure S3.

Fig 5: Repression of the cGAS-STING DNA sensing pathway in ATRX knockdown HFFs.ATRX knockdown HFFs (siATRX) and respective control cells (siC) were treated with (A-C) 0.1 μg/ml poly(dA:dT) and (B+C) DMSO or (D-F) 50 μg/ml 2’3’-cGAMP for 24 h. (A+D) Cells were harvested for Western blot analyses to determine the phosphorylation of IRF3 and TBK1; β-actin served as an internal loading control. Signal intensities were quantified relative to treated siC cells (lane 2). The following antibodies were used: anti-phospho-IRF3 (Ser386) (EPR2346), anti-IRF3 (D6I4C), anti-phospho-TBK1 (Ser172) (D52C2), anti-TBK1 (D1B4), anti-β-actin (AC-15); (A) anti-ATRX (39-f); (D) anti-ATRX (HPA001906). (B+E) Total RNAs were isolated and RT-qPCR was performed to determine IFNB1 induction. Depicted values were calculated from triplicates relative to untreated siC cells using GAPDH as a housekeeping gene and are shown as mean ± SD. One out of (B) three or (E) two independent experiments is shown. Statistical analysis was performed with respective ΔCq-values using a student’s t-test (unpaired, two-tailed); **p<0.01, ***p<0.001. (C+F) Supernatants of stimulated cells were harvested and mixed with HEK-Blue IFN-α/β cells. Type I IFNs in the supernatants were quantitated by measuring SEAP activity produced by HEK-Blue IFN-α/β cells using QUANTI-Blue. Depicted values are derived from triplicates and are shown as mean ± SD. Statistical analysis was performed using a student’s t-test (unpaired, two-tailed); *p<0.05, **p<0.01.