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. 2012 Mar 23;7(3):e32989. doi: 10.1371/journal.pone.0032989

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Fernández-Barral et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Transduction efficiency of M13 primary melanocytes (left panels) and SBcl2 melanoma cell line (right panels) after infection with non-silencing (shNS) or shRNA^mir to HIF1α (shHIF1α) lentivirus at multiplicity of infection of 40 (M13) or 60 (SBcl2). Fluorescence images (40× magnification) show more than 90% GFP-positive cells. (B) Quantitative RT-PCR analysis of HIF1α mRNA levels in M13-shNS, M13-shHIF1α primary melanocytes and SBcl2-shNS, SBcl2-shHIF1α melanoma cell lines. HIF1α mRNA levels are shown relative to control shNS cells after normalization to 18s rRNA. Bars represent average ± standard deviation (SD) (**P<0.01; ***P<0.001). (C) Western blot analysis of extracellular PEDF (PEDF_e) protein levels in conditioned medium (CM), intracellular PEDF (PEDF_i) and HIF1α protein levels in whole-cell extracts from M13-shNS and M13-shHIF1α primary melanocytes incubated under normoxia (21% O₂) or hypoxia (1% O₂) for 16 h and 24 h. β-tubulin was used as loading control. (D) Quantitative RT-PCR analysis of VEGF (left panel) and BNIP3 (right panel) mRNA levels in M13-shNS (filled bars) and M13-shHIF1α (empty bars) primary melanocytes. VEGF and BNIP3 mRNA levels are shown relative to M13-shNS under normoxia after normalization to 18s rRNA. Bars represent average ± SD (*P<0.05; ***P<0.001). (E) Western blot analysis of PEDF_e protein levels in CM, PEDF_i and HIF1α protein levels in whole-cell extracts from SBcl2-shNS and SBcl2-shHIF1α melanoma cell lines incubated under normoxia or hypoxia for 16 h and 24 h. β-actin was used as loading control. (F) Quantitative RT-PCR analysis of VEGF (left panel) and BNIP3 (right panel) mRNA levels in SBcl2-shNS (filled bars) and SBcl2-shHIF1α (empty bars) melanoma cell lines. VEGF and BNIP3 mRNA levels are shown relative to SBcl2-shNS under normoxia after normalization to 18s rRNA. Bars represent average ± SD (**P<0.01; ***P<0.001).