Fig. 4

ERK1/2 inhibition enhances apoptosis of iCCA cells in co-culture system. A Pretreated with ERKi(4 μM), the viability of HuCCT1 and RBE cells with knockdown of ATG7 was detected by CCK-8 assay after co-culturing with activated CD8⁺T cells for 24 h. B Pretreated with ERKi(4 μM) alone or a combination of ERKi and CQ(5 μM), the viability of HuCCT1 and RBE cells was detected by CCK-8 assay after co-culturing with activated CD8⁺T cells for 24 h. C Pretreated with ERKi (4 μM) alone, a combination of ERKi(4 μM) and CQ (5 μM), or knockdown of ATG7, HuCCT1 and RBE cells co-cultured with activated T cells for 24 h were subjected to crystal violet staining. D, F The apoptosis rates of HuCCT1 or RBE cells were detected by Annexin V-APC/7-AAD apoptosis assay using flow cytometry in iCCA cells and activated CD8⁺T cells co-culture system. E, G Fig. E and G are the statistical histogram of D and F. H The apoptosis rates of HuCCT1 or RBE cells after PD-L1 knockdown alone or a combination of ERKi treatment and PD-L1 knockdown were detected by Annexin V-APC/7-AAD apoptosis assay using flow cytometry in co-culture system. Ns, not significant. I PD-L1 protein expression was analyzed by western blot upon PD-L1 knockdown using siRNA. Data shown are mean (SD) from three independent experiments. Ns, not significant. *P < 0.05.**P < 0.01.***P < 0.001