Fig. 4

MMC increased MHC-I expression via activation of p65. a Quantitative real-time PCR data showing MHC-I mRNA levels in NSCLC cells after incubation with MMC for 48 h (0.4 μM). b Western blot analysis of total p65 and phospho-p65 in NSCLC cells after 48-h treatment with MMC (0.4 μM). c Luciferase activity in 293T cells transiently co-transfected with p65 or empty vector and MHC-I promoter or PD-L1 promoter constructs in the presence or absence of p65 expression vector for 48 h. d Western blot analysis showing the upregulation of MHC-I and PD-L1 protein expression by ectopic expression of p65 in NSCLC cells. e, f The mRNA levels of MHC-I and PD-L1 in NSCLC cells incubated with MMC for 48 h (0.4 μM). g, h Western blot analysis showing the knockdown of p65 protein by shRNA; and the MHC-I and PD-L1 expression in the p65 knockdown cells. NTC, no template control. i, j The mRNA levels of MHC-I and PD-L1 in p65 knockdown cells. k, l Western blot analysis of PD-L1 and MHC-I protein expression in p65 knockdown cells after MMC treatment (0.4 μM,48 h). m The PD-L1 and MHC-I protein expression in NSCLC cells treated with TNF-α (20 ng/ml) or MMC (0.4 μM) for 4 8 h. n PD-L1 and MHC-I protein expression in cells treated with MMC (0.4 μM) in the presence or absence of p65 inhibitor (SC-75741 (10 μM)) for 48 h. The H460 and H1299 cells were pre-treated with 10 μM SC-75741 for 2 h. *p < 0.05, **p < 0.01, ***p < 0.001