Stathmin Regulates Hypoxia-Inducible Factor-1 Expression through the Mammalian Target of Rapamycin Pathway in Ovarian Clear Cell Adenocarcinoma
Figure 1
Effects of rapamycin (a) and stathmin knockdown (c) on the phosphorylation of S6K and the expression of HIF-1α and VEGF in RMG1 cells cultured under hypoxic conditions. ((a) and (b)) Cells were treated with 1–20 nM rapamycin and then cultured under normoxic or hypoxic conditions for 5 h. ((c) and (d)) Cells were pretreated for 24 h with control (–) or stathmin (+) siRNAs and then cultured under normoxic and hypoxic conditions for 5 h. (e) Cells were pretreated with or without a PI3K inhibitor, wortmannin (100 nM) for 30 min, and then cultured under hypoxic conditions for 5 h in the presence or absence of EGF. (a, c, and e) Cell lysates were subjected to immunoblot analysis for phosphorylated S6 K (p-S6K), S6K, HIF-1α, phosphorylated Akt (p-Akt), total Akt (Akt), or β-actin. The same blot in each panel was probed, stripped, and then reprobed with each antibody. The levels of β-actin, S6K, and Akt were used as loading controls. Representative data from three independent experiments are shown. ((b) and (d)) Total RNA was extracted and subjected to real-time quantitative RT-PCR to analyze VEGF121 mRNA expression. Data from three independent experiments are shown as ratios of the control level during normoxia, and results are shown as mean ± SEM. * versus normoxia. # versus no treatment.
