Fig. 4: Cholesterol impairs radiotherapy-induced cGAS–STING activation and lovastatin rescues this activation in vitro.

a The expression of p-TBK1, p-IRF3, TBK1 and IRF3 protein extracted from CT26 cells with different treatments (ctrl, cholesterol 50 μM, MβCD 2 mM, IR 6 Gy, IR + cholesterol and IR + MβCD) and detected by western blot. b The relative expression of CCL5, CXCL10 and IFNβ mRNA extracted from CT26 cells was detected by qPCR. c The expression of p-TBK1, p-IRF3, TBK1 and IRF3 protein was extracted from HCT116 cells with different treatments (ctrl, cholesterol 50 μM, MβCD 2 mM, IR 6 Gy, IR + cholesterol and IR + MβCD) and detected by western blot. d The relative expression of CCL5, CXCL10 and IFNβ mRNA extracted from HCT116 cells was detected by qPCR. e, f The expression of p-TBK1, p-IRF3, TBK1 and IRF3 protein extracted from CT26 cells and HCT116 cells with different treatments (ctrl, lovastatin 10 μM, IR 6 Gy and IR + lovastatin) was detected by western blot (e) and quantitative anslysis (f). g, h, Confocal fluorescence microscopy was conducted on CT26 (g) and HCT116 (h) cells with different treatments. The cells were labeled with DAPI (blue) and p-TBK1 (green) or p-IRF3 (green). i The levels of IFN-β and IFN-γ in the supernatant of co-cultures of MC38-OVA cells and OT-1 mouse spleen cells as well as HCT116 cells and human PBMCs were measured using ELISA. The ratio of immune cells to tumor cells is 10:1. The co-cultures were maintained for 36 h. j LDH release assay was performed using the supernatants from co-cultures of MC38-OVA cells and OT-1 mouse spleen cells as well as HCT116 cells and human PBMCs. The ratio of immune cells to tumor cells is 10:1. The co-cultures were maintained for 36 h. Scale bar, 5 μm. **P < 0.01; *P < 0.05; ns, not significant.