Fig. 7

Ubiquitination of BRAF serves to maintain BRAF oncogenic function in melanoma cells. a Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from WM1346 cells stably expressing EV, WT-BRAF, or the 5KR-BRAF mutant. The stable cell lines were further infected with the indicated lentiviral vectors to deplete endogenous BRAF. *The BRAF cDNA used in this figure was mutated to confer resistance to the shBRAF lentiviral vector. b IB analysis of anti-BRAF immunoprecipitates (IP) BRAF-depleted WM1346 cells stably expressing WT-BRAF or the 5KR-BRAF mutant. c WM1346 cells generated in (a) were subjected to cell proliferation assays. Cell viability was calculated as mean ± SD (n = 3) from three independent experiments. *P < 0.05; Student’s t test. d WM1346 cells generated in (a) were seeded (300 cells per well) for clonogenic survival assays. e, f WM1346 cells generated in (a) were subjected to Matrigel invasion assays (e). The invaded distance was calculated as mean ± SD (n = 3) from three independent experiments. *P < 0.05; Student’s t test (f). g, h WM1346 cells generated in (a) were subjected to endothelial cell invasion assays (g). The invaded distance was calculated as mean ± SD (n = 3) from three independent experiments. *P < 0.05; Student’s t test (h). Scale bar: 50 μm. i, j WM3918 cells generated in Supplementary Fig. 11a were subjected to clonogenic survival assays without or with M2-differentiated THP1 (i). The colony numbers were counted from three independent experiments. The colony numbers were calculated as mean ± SD (n = 3), *P < 0.05; Student’s t test (j). k, l WM1346 cells generated in (a) were subjected to soft agar colony- formation assays (k). The colony numbers were counted and calculated as mean ± SD (n = 3) from three independent experiments. *P < 0.05; Student’s t test (l). m, n Tumor pictures (m) and the growth curves (n) for the xenograft experiments with the WM1346 cells generated in (a) were inoculated subcutaneously. The visible tumors were measured at the indicated days. Error bars represent ± SEM (n = 6). *P < 0.05; Student’s t test