Suppression of Hypoxia-Inducible Factor 1α (HIF-1α) by Tirapazamine Is Dependent on eIF2α Phosphorylation Rather Than the mTORC1/4E-BP1 Pathway
Figure 3
Effects of TPZ on Akt and the mTORC1 pathway in HeLa cells.
(A) Cells were cultured in hypoxia for 4 h in the presence of the indicated concentrations of TPZ before western-blotting analysis. (B) Immunoprecipitates prepared from the lysates of HeLa cells with Raptor antibody were used in kinase assays with full-length 4EBP1 as the substrate. (C) Immunoblotting was used to detect the phosphorylation of Akt at Ser473 or Thr308 after TPZ treatment. HeLa cells were transfected with siRNAs specifically targeting TSC2 (D) and 4E-BP1 (E) or control siRNAs, as described in the Materials and Methods. Transfected cells were incubated with or without 20 µM TPZ for 4 h under hypoxic conditions. Proteins were detected by western-blot analysis using specific antibodies.