[28]. Experiments were performed to evaluate the impact of bcl-2 overexpression on the expression of these isoforms and their binding to HIF-1a protein. We found that both the hypoxic conditions and bcl-2 protein level of the cells did not modulate the expression of HSP90a and HSP90b proteins (Figure 7A). We then investigated the effect of bcl-2 on the interaction between HIF-1a and HSP90s proteins by immunoprecipitation of HIF-1a protein. As depicted in Figure 7B, Western blot analysis, using antibodies specifically recognizing the isoform a or b, showed that HSP90b, but not HSP90a, forms a complex with HIF-1a protein in bcl-2 overexpressing cells exposed to hypoxia. To further validate the involvement of HSP90 proteins and to confirm the possibility that HSP90b, rather than a isoform, is involved in HIF- 1a stabilization mediated by bcl-2 in hypoxia, HIF-1a protein expression was evaluated in bcl-2 overexpressing cells after transfection with shRNA targeting the a (shHSP90a) or the b (shHSP90b) isoforms. As control, cells were transfected with scramble shRNA vector (shNC). Western blot analysis confirmed the effective knockdown of the expression of each HSP90 target (Figure 7C,D). Moreover, the specificity of each shRNA against HSP90 was demonstrated by the absence of expression modulation of the other HSP90 isoform, verifying that both shRNAs were highly specific for their respective targets. Interestingly, Western blot analysis showed that shHSP90b (Figure 7C), but not shHSP90a (Figure 7D), completely inhibited hypoxic induction of HIF-1a protein in bcl-2 overex- pressing cells. HIF-1a Stabilization by Bcl-2 Discussion The bcl-2 protein is an inhibitor of apoptosis that has been recognized to play an important role also in a wide range of other biological processes, among which autophagy, DNA repair and drug resistance [21,30–32]. Recent studies, including ours, have demonstrated that bcl-2 also promotes tumour progression and angiogenesis of different tumour histotypes [13,16,33,34]. In this context, we have previously demonstrated that under hypoxic conditions the overexpression of bcl-2 in tumour cells is able to increase tumor angiogenesis enhancing the secretion of the pro- angiogenic factor VEGF, through the induction of HIF-1a protein expression and HIF-1 transcriptional activity [12,13]. In the present study, we investigated the mechanism by which bcl-2 regulates HIF-1a protein expression in M14 melanoma cells under conditions strictly dependent on oxygen availability, such as hypoxia and high cell density. We demonstrated that HIF-1a protein is required for bcl-2-induced VEGF expression under hypoxia by using a small interference approach. Moreover, we confirmed the capability of bcl-2 to modulate VEGF expression in several melanoma cells. We showed that also in high cell density conditions, which create a local pericellular hypoxic microenvi- ronment, bcl-2 overexpression determines an increase of HIF-1a protein expression and HIF-1 transcriptional activity, similar to the ones obtained in hypoxia. Alternatively, bcl-2 is not able to cooperate with insulin or EGF to induce HIF-1a protein expression under normoxia, highlighting that the capacity of bcl- Figure 7. HSP90b is the mediator of HIF-1a induction by bcl-2 under hypoxic conditions. (A) Western blot analysis of HSP90a and HSP90b protein expression in M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia or to normoxia for 24 h. (B) Analysis of HSP90a/HIF-1a and HSP90b/HIF-1a interactions in the cells exposed to hypoxia for 24 h. Protein extracts were immunoprecipitated (IP) with anti-HIF-1a and then Western blot analysis was performed using anti-HSP90a and anti-HSP90b antibodies. (C,D) Western blot analysis of HIF-1a, HSP90a and HSP90b protein expression in bcl-2 stably overexpressing cells transiently transfected with short hairpin construct targeting HSP90b (shHSP90b), HSP90a (shHSP90a) or with control vector (shNC) and exposed to hypoxia or to normoxia for 24 h. (A,C,D) b-actin protein amounts are used to check equal loading and transfer of proteins. (A–D) Western blot analyses representative of two independent experiments with similar results are shown. doi:10.1371/journal.pone.0011772.g007 PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11772