HIF-1a Stabilization by Bcl-2 Figure 1. bcl-2 modulation regulates HIF-1a protein expression in conditions strictly dependent on oxygen avaibility. (A) Western blot analysis of HIF-1a and bcl-2 protein expression in total extracts of M14 cells transfected with siRNA targeting bcl-2 mRNA (si-bcl-2) or with a control scrambled si-RNA (si-contr) and then exposed to normoxia or hypoxia for 24 h. (B) Western blot analysis of HIF-1a and HIF-1b protein expression in total extracts of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) cells plated under low (sparse) or high (dense) cell density conditions, or cultured under normoxia for 4 days or under hypoxia for 24 h. Western blot analysis of HIF-1a and HIF-1b protein expression in total extracts of the cells plated under high cell density conditions and (C) exposed to 24 h shaking or (D) cultured with different volumes of medium. (E) Western blot analysis of HIF-1a and HIF-1b protein expression in total extracts of cells exposed to Insulin (100 nM) or Epidermal Growth Factor (EGF, 20 ng/ml) for 24 h. (A–E) b-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown. doi:10.1371/journal.pone.0011772.g001 involvement of bcl-2 in the regulation of HIF-1a protein synthesis using [35S]-labeled methionine and cysteine in pulse analysis. As shown in Figure 2B, HIF-1a protein synthesis rate was almost identical in control cells and bcl-2 overexpressing clones, indicating that bcl-2 does not affect HIF-1a protein synthesis. Therefore, the potential role of bcl-2 in the regulation of HIF-1a protein turnover was analyzed. As depicted in Figure 2C (left and right panels), a time-dependent decrease of HIF-1a protein level was observed after treatment with the protein synthesis inhibitor Cyclohexamide (CHX) following hypoxia exposure, both in control cells and bcl-2 transfectants. Particularly under CHX exposure for 60 min, the HIF-1a protein was still well detectable in bcl-2 transfectants while weakly in the control cells. Indeed, bcl-2 overexpression increased the HIF-1a half-life from 1565 min to 4565 min under hypoxic conditions (Figure 2C). Similar results were obtained evaluating the effect of bcl-2 on HIF-1a half-life in high cell density conditions, where the HIF-1a protein half-life was about 20610 min in control cells, and increased to 4065 min in bcl-2 transfectants (Figure S3). We confirmed these results performing pulse-chase experiment, in which a pulse with [35S]-labeled methionine and cysteine was followed by a chase time of varying length (ranging from 15 to 60 min). As shown in Figure 2D, HIF-1a degradation rate was higher in control cells compared to bcl-2 transfectants, in fact after 45 min of chase, the HIF-1a protein was still well detectable in bcl-2 PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11772