Figure 5.

CK1γ2 suppresses cell growth through the Akt signaling pathway by degrading IRS4 in osteosarcoma cell lines in vitro and in vivo. (A) U2OS/MTX300 cells stably infected with vectors containing control and CK1γ2-WT were stimulated to 100 ng/μL IGF-1 after serum starvation for the indicated times and then analyzed by Western blotting. (B) U2OS/MTX300 cells stably expressing control vector or CK1γ2-WT were infected with vectors containing IRS4-WT and then analyzed by Western blotting. (C) U2OS/MTX300 cells were stably infected with the indicated plasmids, as shown in (B), and cell proliferation was measured using MTT assays at the indicated times (n = 6). The dots represent the mean, while the bars indicate the SEM. *, P< 0.05 and ***, P< 0.001. A two-tailed Student's t-test was used for statistical analysis. (D) U2OS/MTX300 stable cell lines generated in (B) were cultured in 6-well plates for 14 days, and representative photographs are shown. (E) U2OS/MTX300 cells that stably expressing IRS4-WT or -S859A were transfected with vectors containing control or CK1γ2-WT and then analyzed by Western blotting. (F) U2OS/MTX300 stable cell lines generated in (E) were cultured in 6-well plates for 14 days, and representative photographs are shown. (G) The proliferative capacities of U2OS/MTX300 stable cell lines generated in (E) were measured using MTT assays at the indicated times (n = 6). The dots represent the mean, while the bars indicate the SEM. *, P< 0.05 and ***, P< 0.001. A two-tailed Student's t-test was used for statistical analysis.(H-I) Nude mice were subcutaneously inoculated with the U2OS/MTX300 stable cell lines generated in (A) (2 × 10⁶ cells per mouse) and were allowed to grow for 24 days(n=8 per group). The tumor volumes were measured every 2 days. At day 24, the xenografts were excised from the mice and weighed. Bars represent SEM.*, P<0.05 and ***, P<0.001. A two-tailed Student's t-test was used for statistical analysis.