Fig. 2: Expression of ARF1 R99C induces a STING-dependent type I IFN response.
From: ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling
a ISRE promoter activity by SEAP reporter in 293-Dual-hSTING-R232 cells transiently expressing FLAG-tagged ARF1 WT, R99C, Q71L or T31N on (32 h post transfection and normalised to cell viability). Dots represent mean of n = 3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of whole-cell lysates (WCLs) stained with anti-FLAG. b Area under the curve analysis of the data in (a). c ISRE promoter activity by Firefly luciferase (Fluc) quantification in HEK293T cells transiently expressing STING (+STING) or empty vector (-STING) and FLAG-tagged ARF1 WT and R99C (32 h post transfection and normalised to GAPDH-Renilla luciferase). Dots represent mean of n = 3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained by anti-FLAG. d, e ISRE (d) or NF-κB (e) promoter activity in A549 Dual cells transduced with ARF1 WT and R99C. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h) served as positive controls. Bars represent mean of n = 3 ± SEM (biological replicates). Lower panels: Corresponding immunoblots of WCLs stained by anti-FLAG, anti-STING and anti-GAPDH. f Exemplary immunoblot of HEK293T WCLs transiently expressing indicated ARF1 constructs, cGAS and STING. Blots were stained with anti-pTBK1, anti-TBK1, anti-IRF3, anti-FLAG and anti-GAPDH. g Quantification of the pTBK1 band intensities in (f) normalized to TBK1. Bars represent mean of n = 4 (vector, WT), n = 3 (R99C, Q71L, T31N) ± SEM (biological replicates). h qPCR of OAS1 mRNA in primary normal human lung fibroblasts transduced to express ARF1 WT and R99C 72 h post transduction. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h). Bars represent mean of n = 3 (cGAMP, IFN-β) and n = 6 (vector, WT, R99C) ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained with anti-FLAG, anti-STING and anti-GAPDH. i Supernatant (SN) transfer of primary fibroblasts from healthy donors (n1, f1) or a patient (AGS460) to 293-Dual-hSTING-R232 cells. IFN-β (100 U/mL, 48 h) and cGAMP (10 µg/ml, 48 h). SEAP (ISRE) activity 48 h post transfer and normalised to cell viability. Bars represent mean of n = 5 (n1) and n = 6 (f1, R99C, cGAMP) ± SEM (biological replicates). See also Supplementary Fig. 2. Statistical analysis was performed using two-tailed Student’s t test with Welch’s correction. *p < 0.05; **p < 0.01; ***p < 0.001. Exact P values and Source data are provided in the Source data file.
