Figure 4.

PAK4 is involved in the stabilization of PD-L1 protein in osteosarcoma cells. (a) Western blot for PAK4 and PD-L1 after knockdown or overexpression of PAK4 in osteosarcoma cells. (b) Western blot for PAK4 and PD-L1 in osteosarcoma cells after treatment with PAK4 inhibitor KPT9274 (1 µM for 48 h). (c) Quantitative reverse-transcription polymerase chain reaction for PAK4 and PD-L1 after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. (d) Immunofluorescence staining of U2OS and KHOS/NP osteosarcoma cells after knockdown or overexpression of PAK4. The cells were incubated with primary antibodies for PAK4 and PD-L1. Thereafter, the slides were incubated with Alexa Fluor 488 anti-mouse IgG (green) or Alexa Fluor 594 anti-rabbit IgG (red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Images were taken with a Zeiss LSM 880 with Airyscan confocal microscope. (e) U2OS and KHOS/NP osteosarcoma cell lysates were immunoprecipitated with PAK4 or PD-L1 and immunoblotted with PAK4 and PD-L1. (f) U2OS cells were transfected with control vector or shRNA for PAK4 and treated with 30 µmol/L cycloheximide or 30 µmol/L MG132 for 0.5 to 2.0 h. Thereafter, protein lysates were immunoblotted with PD-L1 and GAPDH. (g) Total protein lysate from U2OS cells transfected with empty vector or shRNA for PAK4 and treated with 30 µmol/L MG132 for two hours was immunoprecipitated with PD-L1 and immunoblotted with anti-ubiquitin antibodies. ** p < 0.001; ns, not significant; CHX, cycloheximide; EV, empty vector; EVs, empty vectors; IP, immunoprecipitation; PAK4-OE, vector for wild-type PAK4; shControl, control vector for shRNA; shPAK4, vector for shRNA for PAK4; WB, western blot.