Fig. 8

CircSLCO1B3 promotes ICC immunosuppression via suppressing protein stability of PD-L1 to alleviate CD8⁺ T cell activity. a The schematic diagram of the trans-well coculturing system. b, c qPCR was conducted to determine the mRNA levels of IFN-γ, TNF-α, IL-4 and IL-10 in CD8⁺ T cells. d FCM was employed to determine the apoptosis ratio of CD8⁺ T cells in the co-culturing system. e PD-L1 protein levels were detected by western blotting after depleting or increasing circSLCO1B3 in ICC cells. f FISH and IF images of circSLCO1B3 and PD-L1 in ICC tissues (circSLCO1B3-FISH: green spots, PD-L1-IF: red spots, DAPI: blue spots) (magnification, × 200, scale bar, 50 μm). g RIP assays were executed to explore the enrichment of circSLCO1B3 by the anti-PD-L1 antibody. h CircRNA pull-down was executed in ICC cells to determine the expression levels of PD-L1 by the circSLCO1B3 probe. i Western blotting was performed at indicated time points to detect the protein levels of PD-L1 in HuCCT1 cells transfected with circSLCO1B3 siRNA after using cycloheximide. j The expression of PD-L1 protein in ICC cells with or without circSLCO1B3 siRNA after treatment with MG-132 by western blot. k Images of xenograft tumors of each group for homologous tumor transplantation (n = 10). l, m IHC staining showed the expression levels of PD-L1, SPOP, and CD3 in tumors from different groups. Scale bar, 50 μm. Mean ± SEM. Student's t-test. *P < 0.05, 0.001 < ** P < 0.01, *** P < 0.001