Fig 2: Cisplatin induced dsDNA and the releasing of chromatin bound cGAS may function as an important role in enhancing STING signal. (A) Basic transcription levels of cGAS in bladder cancer cell line on CCLE database. (B) Relative transcriptional expression of cGAS in different T stages (Normal N = 68, Ta or T1 N = 126, and T2 or more N = 62) in Lee bladder cohort. (C) Western blotting of subcellular distribution of cGAS, Rad51 GAPDH, and Histone H3 proteins. GAPDH was used as cytosolic control protein, and histone H3 was used as nuclear control proteins. T24 and TCCSUP cell treated with 2 μg/mL cisplatin or PBS at 0 h, 8 h, 16 h, 24 h time points. (D) Immunofluorescence of expression and distribution of Rad51 and γ-H2A.X proteins in T24 and TCCSUP cell lines treated with μg/mL cisplatin or PBS for 24 h. DDP, cisplatin. Scale bars represent 10 μm. (E) Immunofluorescence of cGAS subcellular distribution of T24, TCCSUP, MB49 and UMUC-3 cell lines. Scale bars represent 5 μm. (F) Immunofluorescence of cGAS subcellular distribution of T24 and TCCSUP cell lines treated with 2 μg/mL cisplatin or PBS. Scale bars represent 5 μm. (G) Western blotting of subcellular distribution of cGAS, Rad51, γ-H2A.X, GAPDH and Histone H3 proteins. GAPDH was used as cytosolic and nuclear soluble control protein, and Histone H3 was used as chromatin bound control proteins. T24 and TCCSUP cells were treated with 2 μg/mL cisplatin or PBS at 0 h, 8 h, 16 h, 24 h time points. DDP, cisplatin, N.S., p > 0.05, *** p < 0.001.

Fig 3: STING targeting siRNAs and STING specific inhibitor H151 significantly reversed cisplatin related immune effects. (A) Knockdown efficiency validation of STING in T24 and TCCSUP cell lines. GAPDH was used as control protein. (B) T24 and TCCSUP cell lines were treated with non-targeting (Scramble, Scr) or two STING-targeting siRNA (si2 and si3) for 48 h before treatment with 2 μg/mL cisplatin for 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, respectively. IFN-β mRNA and IL-6 mRNA were then analyzed by qRT-PCR. (C) Western blotting results of key proteins involved in cGAS-STING pathway in T24 and TCCSUP cell lines treated with non-targeting (Scramble, Scr) or two STING-targeting siRNA (si2 and si3) for 48 h before treatment with 2 μg/mL cisplatin for 0 h, 8 h, 16 h, 24 h, respectively. GAPDH was used as control protein. (D) T24 and TCCSUP cell lines were co-treated with DMSO or H151 and 2 μg/mL cisplatin for 0 h, 4 h, 8 h, 12 h, 20 h, 28 h, respectively. IFN-β mRNA and IL-6 mRNA were then analyzed by qRT-PCR. (E) Western blotting results of key proteins involved in cGAS-STING pathway T24 and TCCSUP cell lines co-treated with DMSO or H151 and 2 μg/mL cisplatin for 0 h, 8 h, 16 h, 24 h, respectively. GAPDH was used as control protein. * p < 0.05, ** p < 0.01.

Fig 4: Activation of cGAS-STING suppressed bladder cancer in cisplatin-treated C57 mice. (A) Establish of bladder cancer transplantation models in C57 mice, left. Tumor volumes of different groups, right. (B–E) Infiltration percentages of (B) CD8+, CD45+ T cells, (C) CD11b+, MHCⅡ+ DCs, (D) F4/80+, CD11c+ Macrophage, (E) CD3+, CD45+ cells in MB49 transplantation tumors. (F–J) CYBERSORT predicted infiltration percentages of CD8+ T cells (F), DCs activated cells (G), DCs resting cells (H), Macrophages M1 cells (I), and Macrophages M2 cells (J) in response group (n = 50, CR or PR) was significantly higher than non-response group (n = 30, PD or SD) after cisplatin-based chemotherapy. * p < 0.05, ** p < 0.01, *** p < 0.001. DDP, cisplatin.

Fig 5: Treatment with senolytic drug ABT263 reduces senescence in males, but not females, following rmTBI. Western blot analysis revealed that ABT263 treatment 1 week after injuries followed by sacrifice 1 week later (total of 2 weeks post-injuries) significantly reduced expression of p21 in male rmTBI mice (A) (p = 0.02, two-way ANOVA), which was significantly increased by injury (p = 0.0003, main effect of injury, two-way ANOVA). p16 was also significantly increased by injury (p = 0.02, main effect of injury, two-way ANOVA) in the males (A) and was only partially reduced by ABT263 treatment, although this did not reach statistical significance. In contrast, females showed no significant differences in p21 protein levels by this timepoint (B). Interestingly, p16 protein levels significantly increased with ABT263 treatment in female mice (p = 0.03, main effect of treatment, two-way ANOVA). ABT-263 treatment did not significantly reduce markers of DNA damage nor cGAS-STING signaling (C) in male mice, indicating the remaining presence of DNA damage and inflammatory signal. Error bars represent standard deviation with n = 4 per group. Statistical significance is marked as *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.