RND3 downregulation participates in the invasion of melanoma cells insensitive to BRAF inhibition. A) Immunoblot of lysates from cells treated 48 h with 0.5 μM SB-590885, 0.5 μM PLX-4720 or DMSO using antibodies directed toward RND3 (05-723 from Upstate Biotech Inc.) and total ERK2. B-F) Inducible expression of myc-tagged wild-type RND3 in WM793 melanoma cells cultured in complete medium ± doxycycline (Dox) in the absence or presence of PLX-4720. B) Western blot of cell lysates using antibodies specific for myc-tag, RND3, phos-ERK1/2 and ERK2. C) The viability of cells, treated as in (A), cultured four days was monitored by Toludine blue staining, graphed are the results from one experiment performed in triplicate. D) Cells, treated as in (A), plated in serum-free medium into the upper well of a Boyden migration chamber pre-coated with fibronectin + collagen mixture (10 μg/ml). The lower well contained complete medium, to stimulate cell migration. Sixteen hours later, cells that migrated to the insert bottom were labeled with Hoescht Dye and counted by fluorescent microscopy. E) Micrographs depicting invasive outgrowth of spheroids, treated as indicated, embedded inside a 3-D collagen gel. F) Quantitation of the number of spheroids that contained invasive cells, as depicted in (E). All experiments were performed in triplicate. The graphs presented represent mean ± SD, statistical significance determined using Student's t-test and P-values < 0.05 considered significant (*).