Fig. 4

Podoplanin silencing enhances phosphorylation of Ezrin, Radixin and Moesin (E/R/M) proteins in TPC1 and BcPAP thyroid cancer cells. a. RT-qPCR analysis of E/R/M mRNA levels 48 h after transfection with siPDPN or control siNeg. The results were normalized to the 18S rRNA levels. The bars represent the average fold change in transcript levels in cells transfected with siPDPN compared to siNeg-transfected control cells. Data are presented as means ± standard errors of the mean (SEM) of results from at least three independent experiments. b. Western blot analysis of phospho-E/R/M protein levels 48 h after transfection. Total protein extracts were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with specific antibodies. Beta-actin was used as a loading control. The results are representatives of three independent experiments. c. Immunofluorescent staining of podoplanin (red) and phospho-E/R/M (green) proteins in BcPAP (right panels) and TPC1 cells (left panels) transfected with siPDPN or control siNeg. Cells were co-stained with the anti-PDPN mouse antibody D2–40 and anti-phospho-E/R/M rabbit antibody followed by the DyLight549-conjugated anti-mouse secondary antibody (red) and FITC-conjugated anti-rabbit secondary antibody (green), and counterstained with DAPI (blue). Magnification: 630x