Statistical Analysis Differences between groups were analyzed with a two-sided paired or unpaired Student’s t test by use of GraphPad Prism 3.00 (GraphPadSoftware, San Diego, CA). Results were considered to be statistically significant if p,0.05. Experiments were usually repeated three times unless indicated otherwise. Supporting Information Figure S1 HIF-1a protein is required for VEGF induction by bcl-2 in melanoma cells under hypoxia. (A) Western blot analysis of bcl-2 protein expression in whole extracts and (B) ELISA assay of VEGF protein in conditioned medium in several human melanoma cell lines exposed to normoxia and hypoxia for 24 h, after transient transfection with control (empty) or bcl-2 expressing vector (Bcl-2). (C) Western blot analysis of HIF-1a and HIF-1b protein expression in total extracts and (D) ELISA assay of VEGF protein in conditioned medium in M14 cells stably transfected with control (puro) or bcl-2 expression vector (Bcl2/5) after transfection with siRNA directed against HIF-1a (siHIF-1a) or unrelated control mRNA (siNC) and then exposed to normoxia or hypoxia for 24 h. (A,C) 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. (B,D) Results represent the mean 6 SD of 3 independent experiments performed in triplicate. Fold induction of secreted VEGF protein relative to normoxia. * p,0.01 Found at: doi:10.1371/journal.pone.0011772.s001 (0.98 MB TIF) Figure S2 Bcl-2 cooperates with high cell density conditions to induce nuclear HIF-1a protein and HIF-1 transactivation activity. (A) Western blot analysis of HIF-1a and HIF-1b protein expression in cytoplasmic (Cyto) and nuclear (Nucl) protein extracts of M14 control (puro) and bcl-2 overexpressing (Bcl2/5, Bcl2/37) cells plated under low (sparse) or high (dense) cell density condition. b-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analysis representative of two independent experiments with similar results are shown. (B) HRE transcriptional activity of the cells cultured under sparse or dense conditions. Results represent the mean 6SD of 3 References 1. Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3: 721–732. 2. Liu YV, Baek JH, Zhang H, Diez R, Cole RN, et al. (2007) RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha. Mol Cell 25: 207–217. 3. Jeong JW, Bae MK, Ahn MY, Kim SH, Sohn TK, et al. 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Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS, et al. (2008) NF-kappaB links innate immunity to the hypoxic response through transcrip- tional regulation of HIF-1alpha. Nature 453: 807–811. 10. van UP, Kenneth NS, Rocha S (2008) Regulation of hypoxia-inducible factor- 1alpha by NF-kappaB. Biochem J 412: 477–484. 11. Kaelin WG, Jr. (2008) The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat Rev Cancer 8: 865–873. HIF-1a Stabilization by Bcl-2 independent experiments performed in triplicate. Fold induction relative to sparse condition. * p,0.01 Found at: doi:10.1371/journal.pone.0011772.s002 (0.88 MB TIF) Figure S3 Bcl-2 promotes HIF-1a protein stability in high cell density conditions. Western blot analysis (panel left) and quantification (panel right) of HIF-1a protein expression in total lysates of melanoma control (puro) and bcl-2 overexpressing (Bcl2/ 5, Bcl2/37) cells cultured under high cell density conditions (dense) and then treated with Cyclohexamide (CHX, 50 mg/ml) for the indicated times. b-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analysis represen- tative of two independent experiments with similar results are shown. Densitometric analysis (panel right) of the relative Western blot (panel left) was performed using Molecular Analyst Software and normalized with relative controls depending on the analysis performed. Found at: doi:10.1371/journal.pone.0011772.s003 (0.89 MB TIF) Figure S4 Bcl-2 does not cooperate with hypoxic mimetic compounds to induce HIF-1a protein expression. Western blot analysis of HIF-1a protein expression in total lysates of M14 control (puro) and bcl-2 overexpressing (Bcl2/5, Bcl2/37) cells exposed to desferrioxamine (DFO, 50 mM) or Cobalt Cloride (CoCl2, 100 mM) for 3 h. 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. Found at: doi:10.1371/journal.pone.0011772.s004 (0.39 MB TIF) Acknowledgments We are grateful to Adele Petricca for secretarial assistance and to Tania Merlino for English revision of the manuscript. We thank Dr Sergio Anastasi and Dr Fabrizio Antonangeli for helpful suggestions, and Stefania De Grossi and Carla Ramina for technical assistance. Author Contributions Conceived and designed the experiments: DT DDB. Performed the experiments: DT CG MD. Analyzed the data: DT CG EZ GZ DDB. Contributed reagents/materials/analysis tools: EZ GZ DDB. Wrote the paper: DT DDB. 12. 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