Extended Data Fig. 4: AP-1 depletion boosts STING-dependent immune activation.
From: Clathrin-associated AP-1 controls termination of STING signalling
a, HEK293T cells transfected with FLAG-tagged STINGWT were treated with 2.5 µM diABZI or left untreated for 2 h, immunoprecipitated (IP) with anti-FLAG antibody, and analysed by western blot. b, HeLa cells transfected with NC siRNA or AP-1 siRNA for 3 days were treated with 20nM Baf A1 and 2.5 µM diABZI for 2 h before analysed by western blot. GAPDH was used as a loading control. c, HeLa cells incubated with NC siRNA or AP-1 siRNA for 3 days were infected with HSV-1 (MOI = 5) for 14 h and analysed by western blot. GAPDH was used as a loading control. d, HeLa cGAS/STING double KO cells transfected with FLAG-tagged STINGWT, STINGL364A were treated with 2.5 µM diABZI for 2 h were infected with HSV-1 (MOI=5) for 14 h and analysed by western blot. GAPDH was used as a loading control. e, mRNA levels of IFNB1, IFIT1, IFIT2 and IFIT3 in HeLa cells transfected with NC siRNA or AP-1 siRNA for 3 days and treated with 0.5 μg/mL IVT4 were assessed by RT–qPCR. f, NF-kB and IFN-β luciferase assay in HEK293T cells incubated with NC siRNA or AP-1 siRNA for 3 days, followed by transient expression of STING and stimulation with diABZI (2.5 µM). g, Induction of IFNB1, IFIT1, IFIT2, IFIT3 expression was assessed by RT–qPCR in HeLa cells transfected with NC siRNA or AP-1 siRNA for 3 days and then transfected with 1 μg 90mer. h, Induction of IFNB1, IFIT1, IFIT2, IFIT3 expression was assessed by RT–qPCR in HeLa cells transfected with NC siRNA or AP-1 siRNA for 3 days and then transfected with 1 μM 2’3’-cGAMP. i, Induction of IFIT1 expression was assessed by RT–qPCR in HeLa cells transfected with non-targeting control (NC) siRNA or AP-1σ, AP-1β and AP-1γ siRNA for 3 days and then treated with 1 µM diABZI for 3 h. j, mRNA levels of mouse ifi44 in MEFs cells treated with DMSO or 2 µM H-151 for 3 days were assessed by RT–qPCR. k, mRNA levels of mouse Cxcl10, Isg15, ifi44, ifnb1 in MEFs cells treated with 40 µg/mL DMXAA for 3 h were assessed by RT–qPCR. One representative of three (b,e–k) or at least two (a,c,d) independent experiments is shown. For RT–qPCR experiments, ratios of IFNB1, IFIT1, IFIT2, IFIT3 mRNA versus GAPDH mRNA normalized to the untreated groups of each condition. Mean ± s.d. of three (e–k) technical replicates. P values based on two-tailed Student’s t-tests (e–i,k). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (k). Ratios of target proteins versus loading control normalized to the 0 time point of each condition (b).
