Fig. 1

Hypoxia (0.1%) decreases the expression of PD-L1 in T24 bladder cancer cells. PD-L1 expression decreased in T24 cells after 24 h culture in 0.1% oxygen. IFNγ stimulation increased expression of PD-L1 in 20% O₂, but the IFNγ-driven increase was attenuated in cells grown in 0.1% O₂. Cells were seeded and left to adhere for 24 h before placing into a hypoxia chamber for 24 h and/or 10 ng/ml IFNγ added to the culture media. A) Western blotting shows the change in protein levels of PD-L1 with GAPDH used as a loading control. Three independent experiments were carried out and a representative image is shown. B) Quantification by densitometry analysis was performed using ImageJ by calculating the relative densities of both the loading control and the samples to the control untreated lane. These values were then scaled to the relative density values to find adjusted relative values from three independent experiments. C) 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 four independent experiments normalised to normoxia untreated condition to show the relative fold change. D) qPCR shows changes in mRNA levels relative to the mRNA levels of T24 cells cultured in 20% O₂. Data are the mean ± standard error of the mean (SEM) from three independent experiments plated in triplicate with differences calculated using the delta-delta Ct method relative to the expression of reference gene SDHA. Statistical tests are unpaired t tests performed in GraphPad Prism with p values represented as follows: ns = not significant, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001