Fig. 2. Mitotic SENP3 activates cGAS signaling.

A Real-time PCR was used for the analysis of Ifnb1, Isg15, Il6, Ccl5, and Cxcl10 expression in SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells. The data were normalized to Gapdh internal control and represented as mean with SD (n = 3 independent biological replicates). **p < 0.01, ***p < 0.001. B Cell lysates from SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells were blotted with anti-SENP3, anti-phosph-TBK1(pTPK1), anti-TBK1, anti-phosph-P65(pP65), anti-P65, or anti-actin antibodies. C Real-time PCR was used for analysis of Ifnb1 and Isg15 expression in SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells treated with or without RU.521 for 2 days. The data were normalized to Gapdh internal control and represented as mean with SD (n = 3 independent biological replicates). **p < 0.01. D cGAS in SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells was knocked out by using Cas9/CRISPER system plasmid px459 encoding cGAS gRNAs (left panel). Real-time PCR was used for the analysis of Ifnb1 and Isg15 expression in these cells. The data were normalized to Gapdh internal control and represented as mean with SD (n = 3 independent biological replicates). **p < 0.01. E C57BL/6 mice were subcutaneously injected with cGAS-knockout SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells (1 × 10⁶/mouse). Tumor size was determined once in 3 days in two dimensions. Tumor growth curves represent the mean ± SD. Tumors were harvested and pictured on day 28 post-injection. Tumor weight was also measured immediately after harvest and the data from three independent experiments were shown in the bottom panel. Mean ± SD is indicated. **P < 0.01.