Fig. 3

PD-L1 expression decreases with increasing cell density and hypoxia-induced PD-L1 increase occurs only in high-density cells. As T24 cell seeding density increases, the protein and mRNA expression of PD-L1 decrease in both normoxia and hypoxia conditions (p < 0.001 and p = 0.0011 respectively). When the cells are cultured at higher densities (100% confluence) PD-L1 protein and mRNA expression increases after culture in hypoxia compared with normoxia (p = 0.05576 and p = 0.03721 respectively). Cells were counted and seeded into six-well plates at different densities, left to adhere for 24 h and then incubated for 24 h. A) Flow cytometry shows the change in surface expression of PD-L1. Data are the mean ± standard error of the mean (SEM) of the mean fluorescence intensity of 10,000 viable cells from replicates of three independent experiments normalised to normoxia untreated condition to show the relative fold change. B) qPCR shows changes in mRNA levels relative to the mRNA levels of T24 cells cultured in a 20% O₂ incubator. Data are the mean ± standard error of the mean (SEM) from three independent experiments plated in triplicate and the difference calculated using the delta-delta Ct method relative to the expression of reference gene SDHA. Statistical tests are linear models and ANOVA performed using R and RStudio with p values represented as follows: ns = not significant, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001