A, Migration. M10 and WM278 cells seeded in 12-well/insert plates were transfected with EMMPRIN, VEGFR-2 siRNA or scrambled siRNA (Ctl siRNA) before treatment for 24 h in serum-free medium with Emp (25 µg/ml) or with VEGF (200 ng/mL) used as a positive control. After 24 h of incubation, cells were fixed, stained with Diff Quik and counted under a microscope. Columns, means of three independent experiments carried out in triplicate; bars, SD. *, P<0.05. B, Proliferation. M10 and WM278 transfected with VEGFR-2 siRNA or scrambled siRNA (Ctl siRNA) were plated onto 96-well plates and treated with Emp (25 µg/ml) for 48 hours in serum-free medium. The bioreduction of tetrazolium (MTS) was performed for 4 h at 37°C, and cell proliferation was quantitatively assessed by measuring absorbance at 490 nm. Results are presented as the mean of three independent experiments carried out in triplicate; bars, SD. *, p<0.05. C, Caspase-3/7 activity. Transfected melanoma cell lines with VEGFR-2 siRNA or scrambled control (Ctl siRNA) were seeded onto 96- well plates and treated with Emp (25 µg/ml) for 24 hours in serum-free medium. Apo-one homogeneous Caspase 3/7 buffer containing Z-DEVD-R110 substrate was added to the cells and incubate at room temperature for 4 hours. The caspase-3/7 activity was measured using spectrofluorometer at 530 and 485 nm (excitation and emission wavelength respectively). The data represent means of at least three different experiments; bars, ± SD. * p<0.05.

Left, representative melanoma with lower expression of EMMPRIN. (A) EMMPRIN, (B) VEGFR-2 and (C) HIF-2α staining. Right, representative melanoma exhibiting strong expression of EMMPRIN. (D) EMMPRIN, (E) VEGFR-2, and (F) HIF-2α staining.

A. M10 and WM278 cells were transfected for 24 hours with two different EMMPRIN siRNA (Ambion siRNA) or their corresponding scrambled control siRNA (Ctl siRNA) at 33 nmol/L concentration. EMMPRIN and VEGFR-2 protein expression was evaluated by Western blot analysis of 15 µg cell lysates respectively (actin was used as a loading control; representative blot of 3 independent experiments is shown) and conditioned medium (CM) was harvested for gelatinases and uPA zymographies. B. Immunofluorescence of M10 cells treated with EMMPRIN siRNA and immunostained for EMMPRIN, VEGFR-2, uPA and TIMP-1. C. QRT-PCR analyses of EMMPRIN, VEGFR-2 and TIMP-1 showing the mean ±SD of relative expression to PPIA house keeping gene of at least 3 independent experiments. D. M10 and WM278 cells were incubated with CHO-membranes containing or not EMMPRIN (Ctl and Emp respectively). VEGFR-2 and uPA transcript levels were quantified by qRT-PCR showing the mean ±SD of relative expression to PPIA house keeping gene of at least 3 independent experiments. VEGFR-2 protein expression was evaluated by Western blot analysis of 30 µg cell lysates (actin was used as a loading control; representative blot of 3 independent experiments is shown); bars, SD. *, p<0.05.

A. Melanoma cells were transfected with HIF-2α siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to PPIA housekeeping gene of at least three independent experiments; bars, SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. B. HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ PLA detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.