USP7 stabilizes cGAS and activates the cGAS-STING pathway. (A) MM-231 cells were immunofluorescent stained with HMGA1, EZH2, USP7, and cGAS to observe the co-localization of HMGA1, EZH2, and USP7 with cGAS, Scalebars = 5 μm. (B) MM-231-shUSP7 cells were immunofluorescent stained with cGAS and H3K27me3 to calculate the ratio of cGAS activation. (C) After 48 h of transient transfection of USP7 and cGAS overexpression constructs in HEK-293T cells, the Flag antibody was used for co-IP. Western blotting was used to detect the USP7, cGAS, P-STING, STING, IL-6 level in HEK-293T (C), MM-231-shCtrl/MM-231-shUSP7 (D), MM-231 cells treated with P5091 (10 μM) for 48 h (E), MM-231 cells treated with MG132 (10 μM) for 10 h (F), MM-231-dnMCAK cells treated with P5091 (10 μM) for 48 h (H). (G) HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with MG132 (10 μM) for 10 h. Cell lysates were immunoprecipitated using an anti-HA antibody followed by immunoblotting analysis. Each experiment was repeated at least 3 times. Error bars, mean ± SD. **, p < 0.01.