FIG. 4.

Effect of rapamycin on HIF-1α turnover. (A) Proteasome inhibition antagonizes the suppressive effect of rapamycin on hypoxia-induced HIF-1α expression. (Left panel) PC-3 cells were incubated for 6 h in 2% FBS before pretreatment for 45 min with solvent vehicle, rapamycin (Rap), or LLnV. Cells were incubated under normoxic conditions (−) or hypoxia (H). The cells were harvested after 16 h, and soluble proteins were resolved by SDS-PAGE. (Right panel) PC-3 cells were precultured for 16 h in 0.1% FBS prior to the indicated drug treatments and were then stimulated for 6 h with 150 μM CoCl₂ (C). The blotted proteins were probed sequentially with α-HIF-1α MAb and α-PLCγ1 antibodies. Expression of HIF-1α protein was quantitated by densitometry and was normalized to the value obtained in the corresponding normoxic control samples. (B) LLnV-induced HIF-1α accumulation. PC-3 cells were treated for the indicated times with 10 μM LLnV under normoxic conditions, in the absence or presence of 100 nM rapamycin. HIF-1α expression was determined by immunoblotting, and the blot was stripped and reprobed with α-PLCγ1 antibody to control for sample loading. The results shown are representative of those obtained in three independent trials. (C) Effect of rapamycin on HIF-1α degradation. Cells were cultured in medium containing 2% serum and exposed to hypoxia overnight. The cells were then treated with CHX to block new protein synthesis, and the indicated samples were concomitantly exposed to 100 nM rapamycin. The cells were maintained under hypoxic conditions for the indicated times, and HIF-1α levels were determined by immunoblotting as described in panel A. PLCγ1 served as a sample loading control as described above.