The effects of PAK4 expression on proliferation and apoptosis of osteosarcoma cells under treatment of doxorubicin. (a,b) MTT proliferation assays (a) and colony-forming assay (b) after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells treated with 0.1 µM doxorubicin. In colony-forming assay, U2OS (3 × 103) and KHOS/NP (3 × 103) cells were seeded in culture plates for one week. (c,d) Western blotting (c) and Annexin V flowcytometric analysis for apoptosis (d) after knockdown or overexpression of PAK4 in osteosarcoma cells under treatment with 0.1 µM doxorubicin. Doxorubicin was applied to osteosarcoma cells 24 h after transfection. * p < 0.05; ** p < 0.001; ns, not significant; EVs, empty vectors; PAK4-OE, vector for wild-type PAK4; shPAK4, vector for shRNA for PAK4; DOX, doxorubicin.