Fig. 10

VEGFA is a miR-126-3p target and its silencing impairs proliferation and invasiveness of dabrafenib-resistant cells. a, b Melanoma cells were transiently transfected with 50 nM pre-miR-126-3p or pre-miR-CTRL and after 72 h of culture, cell supernatants were collected and the amount of VEGF-A quantified by ELISA. Each value represents the arithmetic mean of three (A375R) or four (SK-Mel28R) independent experiments performed with duplicate samples. Bars, SEM. ^**P<0.01 and ^*P<0.05, pre-miR-126-3p vs pre-miR-CTRL. c A375R cells were co-transfected with 100 ng of the indicated vectors and 10 ng of the pRL-null vector encoding Renilla luciferase along with 50 ng of pre-miR-126-3p or pre-miR-CTRL. Firefly luciferase levels were determined 48 h after transfection and normalized to those of Renilla luciferase. Data are expressed in terms of percentage of luciferase activity detected in pre-miR-126-3p-transfected cells with respect to that of pre-miR-CTRL-transfected cells. Each value represents the arithmetic mean of five independent experiments. Bars, SEM. ^*P<0.05, pre-miR-126-3p vs pre-miR-CTRL. d A375R cells (7.5x10⁴) were plated into 6 cm-dishes and 18 h later transiently transfected with 50 nM siVEGFA or siCTRL. After 72 h of culture the cells were detached and counted. Each value represents the arithmetic mean of three independent experiments performed with duplicate samples. Bars, SEM. ^**P<0.01, siVEGFA vs siCTRL. e A375R cells were plated and treated as in (d) and 72 h after transfection assayed for ECM invasion. Left panel, number of invaded cells per microscopic field. Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. Bars, SEM. ^**P<0.01, siVEGFA vs siCTRL. Right panel, representative images of polycarbonate filters with invaded melanoma cells.