Fig. 4.

Inhibition of HIF-1 by digoxin is independent of mTOR and Na⁺/K⁺ ATPase α1 subunit. (A) PC3 (Left) and P493 (Right) cells were cultured at 20% or 1% O₂ for 24 h in the presence of vehicle (V), 100 nM digoxin (D), or 25 nM rapamycin (R) and whole cell lysates were subjected to immunoblot assays with antibodies specific for HIF-1α, phosphorylated ribosomal protein S6 (P-RPS6), total RPS6, or β-actin. (B) RNA was isolated from untreated 293T cells (Parental) or cells transfected with empty vector (EV) or expression vectors encoding two different short hairpin RNAs against the Na⁺/K⁺ ATPase α1 subunit (sh811, sh2293). The levels of α1 subunit mRNA (mean ± SD) were quantified by real-time RT-PCR. *, P < 0.05 vs. Parental or EV (Student's t test). (C) 293T cells were transiently transfected with EV or vector encoding sh811 or sh2293 and treated with vehicle (−) or 100 nM digoxin (+) at 1% O₂ for 24 h. Whole cell lysates were subjected to immunoblot assays for HIF-1α and β-actin.