Loss of Trp53 and Rb1 in Ctsk-expression activates YAP/TAZ signaling. (A) Schematic presentation of primary osteosarcoma cells from Ctsk-Cre;Trp53f/f/Rb1f/f mice. (B) Western blot analysis of YAP and TAZ expression in primary osteosarcoma cells and normal cortical bone cells. (C) Representative images of immunofluorescent staining of YAP/TAZ in the primary osteosarcoma cells and normal cortical bone cells as indicated. Scale bars, 10 μm. (D) The normal cortical bone cells or primary osteosarcoma cells with/without 2 μmol VP were co-transfected with 8xGTIIC and pRL-TK plasmids (internal control) as indicated, respectively. After transfection of 48 h, the luciferase activities were identified by the Dual-Luciferase Assay Kit. (E) The primary osteosarcoma cells were treated by 2 μmol VP or DMSO (control) for 48 h, the protein levels of YAP and TAZ were identified as indicated. (F) qRT-PCR analysis of YAP/TAZ target genes CYR61 and CTGF in the normal cortical bone cells or primary osteosarcoma cells cultured with/without 2 μmol VP for 48 h as indicated. Error bars were the means ± SEM from three independent experiments. * p < 0.05. ** p < 0.01, *** p < 0.001.