Fig. 5

Restoration of p53 knockdown-associated JAK2 downregulation improves IFN-ɣ-inducible PD-L1 expression. a Immunoblot of three melanoma cell lines containing either an inducible TP53-targeting or a scr shRNA expression vector. IFN-ɣ treatment was for 48 h. b, c Linear regression analyses of JAK2 mRNA with CD274 mRNA (b; n = 347) or PD-L1 protein (c; n = 262). d M19-MEL and UACC-62 cells containing an inducible TP53-targeting shRNA vector were treated for 6 days with doxycycline with the last 2 days either in absence or presence of IFN-ɣ. After RNA isolation and cDNA generation, real-time quantitative PCR was performed for determination of TP53, JAK2 and CD274 mRNA expression. Relative expressions were calculated by the ΔΔCq method to the respective cell line sample without doxycycline and IFN-ɣ treatment. After log2 transformation, the means + SD of three independent experiments are depicted. Significant differences are indicated by stars (* < 0.05; ** < 0.01; paired t-test). e Two melanoma cell lines containing Doxycycline-inducible p53 shRNA were transduced with a JAK2 expression construct. Control cells and JAK2-overexpressing cells were incubated with doxycycline and treated with IFN-ɣ for 48 h as described before. Expression of indicated proteins was determined by immunoblot. ß-tubulin served as a loading control. p < 0.05 is regarded as statistically significant. DOX, doxycycline; scr, scramble; TPM, transcripts per million, ctrl, control. All blots are representative of two individual experiments