Fig. 3

Presence of p53 protein, but not its transcriptional activity determines extent of IFN-ɣ induced PD-L1 expression in melanoma. a Immunoblot for PD-L1 and p53. NSCLC cell lines A549 and H460 served as control; all other are melanoma cell lines. IFN-ɣ treatment was for 48 h. b p53 was rendered transcriptionally inactive by introducing deletions using CRISPR/Cas9 technology and gRNA targeting exons 7 and 9 (LOX-IMVI) or exons 8 and 9 (UACC-62), respectively. Loss of transcriptional activity was determined by expression of GFP-based p53 reporter (left histograms; red: parental cells; blue: cells after CRISPR/Cas9 genome editing). Protein expression of p53 and PD-L1 in the absence or presence of IFN-ɣ for 48 h was determined by immunoblot. c Immunoblot for PD-L1 and p53 in TP53-mutated melanoma cells upon shRNA-mediated p53 knockdown. Cells were treated with IFN-ɣ as their TP53-wildtyp counterparts described in (a). p53 knockdown was achieved by culturing cells in doxycycline for 6 days. d, e Two TP53-wt melanoma cell lines (d) or a TP53-knockout cell line (e) were transduced with doxycycline-inducible p53^L22Q,W23S expression construct. Cells were incubated with doxycycline and treated with IFN-ɣ for 48 h, as described before. Expression of indicated proteins was determined by immunoblot. Arrow (d) indicates ectopic p53 expression. Please note, that for M19-MEL (d) the ectopic p53 expression was so much stronger than the endogenous that, in those samples without doxycycline, the signal for endogenous p53 was too low to be detected. ß-tubulin (a-d) or actin (e) served as loading controls. DOX, doxycycline. All blots are representative of two individual experiments