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

Figure 7. LC3 knock-down prevents downregulation of PEDF by hypoxia in melanoma cells.

Figure 7

(A) Transduction efficiency of SBcl2 melanoma cell line after infection with non-silencing (shNS) or shRNAmir to LC3 (shLC3) lentivirus at multiplicity of infection of 60. Fluorescence images (20× magnification) show more than 90% GFP-positive cells. (B) Quantitative RT-PCR analysis of LC3 mRNA levels in SBcl2-shNS and SBcl2-shLC3 melanoma cell lines. LC3 mRNA levels are shown relative to SBcl2-shNS after normalization to 18s rRNA. Bars represent average ± standard deviation (SD) (***P<0.001). (C) Western blot analysis of extracellular PEDF (PEDFe) protein levels in conditioned medium (CM), intracellular PEDF (PEDFi), HIF1α and LC3 protein levels in whole-cell extracts from SBcl2-shNS and SBcl2-shLC3 melanoma cells incubated under normoxia (21% O2) or hypoxia (1% O2) for 24 h. β-tubulin was used as loading control.