Figure 4

Endogenous RHOA depletion antagonizes the invasion of melanoma cells that are insensitive to BRAF inhibition. A) Lysates generated from cells treated 48 h with 0.5 μM SB-590885, 0.5 μM PLX-4720 or DMSO, subjected to Western blot analysis using antibodies specific for phosphorylated (Thr18/Ser19) myosin light chain 2 (Cell Signaling #3674) and actin. B) Dox-inducible RHOA shRNA (target sequence: ATGGAAAGCAGGTAGAGTT) melanoma cells were treated ± 0.5 μM PLX-4720 for 48 h. Cell lysate analyzed by Western blot for RHOA (sc418), phos-MLC2, phos-ERK1/2 and ERK2. C) The viability of cells, treated as in (B), monitored by toludine blue staining, representative graph of the results from one experiment performed in triplicate. D) Boyden chamber analysis of cell migration (as described in Figure 3D) following treatments shown in (B). Graph indicates average number of migrated cells ± SD. E) Micrographs of invasive outgrowth associated with inducible RHOA shRNA spheroids in the presence or absence of mutant BRAF inhibition. F) Quantitation (average ± SD) of the number of spheroids, as depicted in (E), that harbor drug refractive cells. All experiments were performed in triplicate. Statistical significance determined using Student's t-test and P-values < 0.05 were considered significant and represented by *.