Figure 4.
MITF regulates PEDF expression in melanoma cell lines. (A) Transduction efficiency of 501mel melanoma cell line after infection with non-silencing (shNS) or shRNAmir to MITF (shMITF) lentivirus at multiplicity of infection (MOI) of 100. Fluorescence images (10x magnification) show more than 90% GFP-positive cells. (B) Quantitative RT-PCR of PEDF, MITF and TYR mRNA levels in 501mel-shNS and 501mel-shMITF melanoma cell lines. PEDF, MITF and TYR mRNA levels are shown relative to control shNS cells after normalization to GAPDH mRNA levels. Bars represent average +/- standard deviation (SD), and statistical significance was determined by Student’s test (***, P < 0.001; ****P < 0.0001). (C) Western blot analysis of extracellular PEDF (PEDFe) protein levels in concentrated conditioned medium (CM), intracellular PEDF (PEDFi) and MITF protein levels in whole-cell extracts from 501mel-shNS and 501mel-shMITF melanoma cell lines. β–tubulin was used as loading control. (D) Transduction efficiency of WM278 melanoma cell line after infection with control (pCDH) and pCDH-HA-MITF (MITF) lentivirus at 100 MOI. Fluorescence images (10x magnification) show more than 90% GFP-positive cells. (E) Quantitative RT-PCR of PEDF, MITF and TYR mRNA levels in WM278-pCDH and WM278-MITF melanoma cell lines. PEDF, MITF and TYR mRNA levels are shown relative to control pCDH cells after normalization to 18s rRNA levels. Bars represent average +/- SD, and statistical significance was determined by Student’s test (**P < 0.01). (F) Western blot analysis of PEDFe protein levels (PEDF band indicated by an arrow) in direct conditioned medium (left), MITF and HA-tag protein levels (right) in whole-cell extracts from WM278-pCDH, WM278-MITF and 501mel melanoma cell lines. β–actin was used as loading control.