Fig. 4

Effects of knockdown and overexpression of TPD52 on growth and apoptosis of SAS cells. a, b siRNA for TPD52 or control siRNA was transfected into SAS cells, and incubated under normoxic conditions for 24 h. Then, the cells were exposed to hypoxia in the presence or absence of 10 μM PX-478. After 0, 1, 2, and 3 d cells were subjected to MTT and caspase 3/7 assays (a), and after 0 and 3 d, total cellular proteins were subjected to western blotting analysis to determine expression of TPD52, HIF-1α, p62, Akt, p-Akt, and β-actin (b). c, d HaloTag-TPD52 or control HaloTag vector was transfected into SAS cells, and incubated under normoxic conditions for 24 h. Then, the cells were exposed to hypoxia in the presence or absence of 10 μM PX-478. After 0, 1, 2, and 3 d, cells were subjected to MTT and caspase 3/7 assays (c), and after 0 and 3 d, total cellular proteins were subjected to western blotting analysis to detect TPD52 (overexpressed and endogenous proteins indicated with arrows with the molecular weight), HIF-1α, p62, Akt, p-Akt, HaloTag, and β-actin (d). For MTT and caspase 3/7 assays, the value at time 0 is designated as “1,” and relative values are shown. The values of 3 d were subjected to ANOVA. *, p < 0.05. Experiments were repeated 3 times