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. 2020 Nov 3;11:5387. doi: 10.1038/s41467-020-19170-5

Fig. 1. The HUSH complex regulates the type I IFN response.

Fig. 1

a Diagram of IFN induction assay: HEK293 cells harbor an integrated IFN reporter expressing destabilized GFP (dscGFP). ISRE: IFN-stimulated response elements; mCMV: minimal CMV promoter. GFP was assessed 6 days post shRNAs. b Western blot and qRT-PCR to assess HUSH depletion day 4 post shRNAs. PPHLN1 mRNA normalized to GAPDH. N = 3 biologically independent samples with technical qRT-PCR duplicates shown. c ISRE-GFP induction 6 days post shRNAs. N = 3 biologically independent experiments. Two-tailed unpaired t tests of shRNAs vs. shControl: p = <0.0001 (shMPP8), p = 0.0372 (shPPHLN1) and p = 0.0466 (TASOR). d shMPP8 was combined with shTASOR or controls as stated. qRT-PCR of endogenous ISG mRNA expression (GAPDH normalized). N = 3 biologically independent experiments with technical duplicates shown. A two-tailed unpaired t test was used to compare shMPP8 + shControl to shMPP8 + shTASOR for CCL5 (p = 0.0021). e shTASOR or shControl-treated cells were treated with IFN-β and ISRE-GFP measured 24 h later. N = 3 biologically independent experiments. P value: 0.0037 (two-tailed paired t test). MFI: mean fluorescent intensity. f QRT-PCR of ISG expression (GAPDH normalization) 6 days post shRNAs (or 3 days in the case of HeLa cells). N = 3 biologically independent experiments with technical duplicates shown. Two-tailed paired t tests were used to compare controls to shMPP8 for CXCL10: p = 0.0473 (HeLa), p = 0.0229 (THP-1s) and p = 0.0084 (HFFs). g IFN bioassay: supernatants from shRNA-treated 293 cells were added to ISRE-GFP reporter 293 cells and GFP expression measured 24 h later. N = 4 biologically independent samples. P = 0.0240 (two-tailed paired t test). h 293 cells containing an IFN-β-destabilized GFP reporter were transduced with the stated shRNAs and GFP measured 6 days later. N = 3 biologically independent samples. P = 0.0005 (two-tailed paired t test). i Supernatants were harvested 6 days post shRNAs and used for an IFN-β ELISA. N = 4 biologically independent samples. P = 0.0023 (two-tailed paired t test). j 293 cells treated with shRNAs in the presence of the JAK/STAT inhibitor Ruxolitinib or DMSO (added from day 1) were harvested for GFP FACS at day 6. N = 4 biologically independent samples. IFN-β  ± Ruxolitinib served as a positive control. P = < 0.0001 (two-tailed unpaired t test). k IFN bioassay: supernatants from shRNA-treated HFFs were added to ISRE-GFP reporter 293 cells and GFP expression measured 24 h later. N = 3 biologically independent samples. Two-tailed paired t test p values: p = 0.0469 (shMPP8), p = 0.0320 (shPPHLN1). All data presented in this figure show mean values ± SD except (b), which depicts mean values ± SEM.