Fig. 2

RelB regulates PD-L1 in PCa cells. a RelB was silenced in PC-3 and DU-145 cells using a lentiviral shRNA specifically targeting endogenic RelB. The NF-κB binding activities in RelB-silenced cells (shRelB) vs. scramble sequence (shCtrl) were measured using an ELISA kit with a standard probe containing the consensus NF-κB element. b mRNA expression profiles of RelB-silenced cells vs. control cells were examined by RNA sequencing. The relative levels of inflammatory and immune responsive transcripts were analysed, as indicated by the heatmap. The signature of CD274 gene expression was indicated in a green box. c-d The levels of RelA, RelB and PD-L1 mRNA and proteins were quantified using RT-qPCR and immunoblots by normalizing to GAPDH. e PC-3 cells were treated with different doses of IFN-γ as indicated. The stimulated PD-L1 mRNA expression was quantified by RT-qPCR with GAPDH normalization. f After IFN-γ treatment, the levels of nuclear RelB and relative cytosolic PD-L1 in PC-3 cells were measured by immunoblots. PCNA served as an internal control for the nuclear fraction, and GAPDH served as a loading control for the cytosolic fraction. g Accordingly, RelB nuclear translocation and relative PD-L1 in the cytosol were confirmed using a confocal microscope. *(p < 0.05) and **(p < 0.01) show significance between the two groups as indicated