Fig. 3: CAD impairs STING dimerization, cGAMP-binding, and Golgi apparatus translocation independent on its nuclease activity.
From: CAD hijacks STING to impair antitumor immunity and radiotherapy efficacy of colorectal cancer
A Immunoprecipitation and western blotting analyses of the effects of CAD on STING dimerization/oligomerization in HEK293T cells. B Immunoprecipitation and western blotting analyses of the effect of CAD on the interaction between STING and cGAMP in HEK293T cells transfected with different amount of CAD. C Representative confocal fluorescence microscopy images of STING-EGFP and cGAMP-Cy5 in indicated cells. Scale bars, 10 μm. D Representative confocal fluorescence microscopy images of STING and Golgi apparatus in indicated cells stimulated with cGAMP. Scale bars, 10 μm. E Immunoprecipitation and western blotting analyses of the effect of CAD on the interaction between STING and TBK1. F Immunoprecipitation and western blotting analyses of the interaction between CAD and the STING (R284S) mutant in HEK293T cells. G Western blotting analyses of p-TBK1, TBK1, p-IRF3, and IRF3 levels in HCT116 and HT29 vector control or Flag-CAD overexpressed cells that transfected with myc-tagged wild type (WT) STING or the R284S mutants at 24 h after irradiation. H Western blotting analyses of p-TBK1, TBK1, p-IRF3, and IRF3 levels in indicated cells at 24 h after treatment of irradiation plus DMSO or H-151 treatment. I qPCR analyses of CCL5, CXCL10, and IFN-β mRNA in indicated cells at 24 h after irradiation with DMSO or H-151 treatment. J Western blotting analyses of p-TBK1, TBK1, p-IRF3, and IRF3 levels in indicated cells at 24 h after treatment of irradiation plus DMSO or RU.521 treatment. K qPCR analyses of CCL5, CXCL10, and IFN-β mRNA in indicated cells at 24 h after irradiation with DMSO or RU.521 treatment. L Immunoprecipitation and western blotting analyses of the interaction between STING and CAD (H260A) mutant in HEK293T cells. M Western blotting analyses of p-TBK1, TBK1, p-IRF3, and IRF3 levels in control cells and the CAD-KO2 cells that reintroduced the wild type (WT) or nuclease activity dead mutant (H260A) of CAD at 24 h after irradiation. N qPCR analyses of CCL5, CXCL10, and IFN-β mRNA in indicated cells at 24 h after irradiation. Error bars represent mean ± SD. P values were determined using two-tailed, unpaired t-test (I, K, and N). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns not significant.
