Fig. 3

SPRED1 loss is selected for in human melanoma cells under continuous BRAF inhibition. (A) Representative pictures at indicated passages (P) of cultures of BRAF-driven A375 human melanoma cells transiently transfected with a vector expressing Cas9 and either a control gRNA or a gRNA targeting SPRED1 and treated with DMSO (vehicle) or 100 nM dabrafenib (Dab). Scale bar, 100 µm. (B) Evolution of frameshift variant allele fraction in cultured A375 human melanoma cells transiently transfected with two independent vectors targeting SPRED1 by CRISPR and treated with either DMSO or 100 nM dabrafenib over five passages. Representative of three independent experiments. (C) Western blot analysis of SPRED1 levels in the cultures described in B. Representative of three independent experiments. (D) Western blot analysis of MAPK pathway activity in A375 human melanoma cells transiently transfected with three independent vectors targeting SPRED1 by CRISPR and stimulated with 0, 10, 30, or 100 nM dabrafenib before any treatment (P0) or after five passages in 100 nM dabrafenib (P5). Representative of three independent experiments.