Fig. 4: TMEM120A promotes STING translocation and downstream signaling.

a, b siRNA-mediated STING knockdown abolished the antiviral function of TMEM120A in U87MG cells. U87MG cells stably expressing luciferase or TMEM120A were transiently transfected with non-targeting siRNA (NT) or siRNA targeting STING for 48 h and infected with ZIKV at MOI of 0.1 for another 48 h. Cells were then harvested for RT-qPCR (a) and immunoblotting (NS1 antibody) (b). c, d TMEM120A promoted STING trafficking from the ER to ERGIC. HEK293T cells were transiently transfected with plasmids expressing STING, GFP-ERGIC53 (green), and HA-FLAG-luciferase or TMEM120A. 48 h posttransfection, cells were fixed and permeabilized for immunostaining using STING (red) antibody and confocal analysis (c). Scale bar: 20 μm. STING-ERGIC53 co-localization was quantified using Pearson’s correlation coefficient method. Cells expressing both STING and ERGIC53 were selected randomly for co-localization analysis by Volocity software (d). ERGIC53 is an ERGIC marker. e siRNA based SEC23A knockdown blocked the antiviral function of TMEM120A in U87MG cells. U87MG cells stably expressing Luciferase or TMEM120A were transfected with NT control siRNA or siRNA targeting SEC23A for 48 h and infected with ZIKV at an MOI of 0.1 for another 48 h. Cells were harvested for RT-qPCR. f, g siRNA based SEC23A knockdown decreased the translocation of STING from ER to ERGIC in HEK293T cells. HEK293T cells were transfected with NT or siRNA targeting SEC23A for 48 h, and then co-transfected with plasmids expressing STING, GFP-ERGIC53, and HA-FLAG tagged TMEM120A for another 48 h. Cells were fixed and permeabilized for immunostaining using STING (red) antibody and confocal analysis (f). Scale bar: 20 μm. STING-ERGIC53 co-localization was quantified using Pearson’s correlation coefficient method. Cells expressing both STING and ERGIC53 were selected randomly for co-localization analysis by ImageJ software (g). h, i TMEM120A promoted TBK1 and IRF3 phosphorylation in HEK293T cells. HEK293T cells stably expressing STING were transiently transfected with HA-FLAG tagged luciferase or TMEM120A. 40 h posttransfection, these cells were stimulated with 2′, 3′-cGAMP (2 μg/mL) for the indicated time (0, 3, 6 h). Cells were collected for immunoblotting to detect the activation of TBK1 (h) and IRF3 (i) using phospho-TBK1 (p-TBK1), TBK1, p-IRF3, and IRF3 antibodies. LE, long exposure. SE, short exposure. Ratio: p-TBK1/TBK1 or p-IRF3/IRF3. j TMEM120A overexpression promoted type I IFN response in U87MG cells. U87MG cells stably expressing luciferase or TMEM120A were infected with ZIKV at an MOI of 0.1 for 48 h. Cells were harvested for RT-qPCR to detect the mRNA level of IL6, IL8, IFIT2. k shRNA based TMEM120A knockdown decreased type I IFN response in U87MG cells. U87MG cells stably expressing NT shRNA or TMEM120A shRNAs (shRNA-1, shRNA-2) were infected with ZIKV at an MOI of 0.1 for 48 h. Cells were harvested for RT-qPCR to detect the mRNA level of IL6, IL8, IFIT2. l TMEM120A overexpression promoted type I IFN response in RAW 264.7 cells. RAW 264.7 cells were transiently transfected with luciferase or TMEM120A and then infected with HSV-1 at an MOI of 1 for 6 h. Cells were harvested for RT-qPCR to detect the mRNA level of Ifnb1 and other Isgs. Cellular ZIKV RNA level and gene mRNA level were normalized to the internal control GAPDH. RT-qPCR data in (a, e, j, k, l) represent the mean ± SEM (n = 3 independent experiments). Quantification of immunostaining data in (d, g) represent the mean ± SEM (n = 25 cells per group). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired, two-sided Student’s t-test). ns: not significant. Exact P values and statistical parameters are provided in Source Data File.