Impaired ISG induction in IRF9-deficient cells. (A) Transcription levels of MX1, IFIT1, IFIT3, and CXCL9 assessed by qRT-PCR on F-SV40 cells treated with 1,000 U/ml of IFN–α2b, -β, or –γ for 2 h. Cells were from three healthy controls (C1, C2, and C3), an IRF9-deficient patient (IRF9−/−), her mother (IRF9+/−), and STAT1-deficient (STAT1−/−), STAT2-deficient (STAT2−/−), IRF7-deficient (IRF7−/−), and IFNGR1-deficient (IFNGR1−/−) patients. Representative results of four independent experiments are shown. (B and C) WB of MX1 and IFIT3 on F-SV40 (B) or B-LCL (C) cells treated with 1,000 U/ml of IFN–α2b for various time points. GAPDH was used as a loading control. Representative results of three independent experiments are shown. (D) Transcription levels of MX1, IFIT1, IFIT3, and CXCL9 assessed by qRT-PCR of B-LCL cells treated with 1,000 U/ml of IFN–α2b, -β, or –γ for 2 h. Cells were from three healthy controls (T1, T2, and T3), an IRF9-deficient patient (IRF9−/−), her mother (IRF9+/−), and STAT1-deficient (STAT1−/−), STAT2-deficient (STAT2−/−), IRF7-deficient (IRF7−/−), and IFNGR2-deficient (IFNGR2−/−) patients. Representative results four independent experiments are shown. (E) Transcription levels of MX1, IFIT1, and CXCL9 assessed by qRT-PCR in F-SV40 cells from a healthy control (C1), P’s mother (IRF9+/−), and P (IRF9−/−) stably transfected with luciferase as a control (Luc) or indicated IRF9 variants (WT: WT IRF9, green: reported loss-of-function variants, blue: variants found in-house, red: patient variant). Cells were stimulated with 1,000 U/ml of IFN-α2b, -β, or -γ for 2 or 8 h. Representative results of four independent experiments are shown. (F) Similar to E, qRT-PCR analysis of MX1, IFIT1, and CXCL9 expression in parental HT1080 cells and U2A cells. Cells were stimulated with 1,000 U/ml of IFN-α2b, -β, or -γ for 2 or 8 h. Representative results of three independent experiments are shown.