The transformed phenotype of RET/PTC3 thyroid cells requires the integrity of the Y1062-RAS-BRAF-ERK pathway. (A) PC cells were transfected with the indicated plasmids or the empty vector and either selected in neomycin-containing medium or left in the absence of 6H. Three weeks later, colonies were stained with crystal violet and counted. The ratio of neomycin-resistant clones to 6H-independent colonies was calculated. The results of 3 independent experiments performed in duplicate ± SD are reported [the number of 6H-independent colonies induced by HRAS(V12) was set at 100]. Inset: Protein lysates (50 μg) underwent Western blotting with the indicated antibodies. cyc D1, cyclin D1. (B) Matrigel invasion of parental and transformed PC cells. Where indicated, in PC RET/PTC3 cells, suppression of endogenous BRAF was obtained by transfection of RNA interference against BRAF, and ERK inhibition was achieved by U0126 treatment. Cells were seeded in the upper chamber of 8-μM-pore transwells and incubated for 24 hours. Thereafter, filters were fixed and stained. The upper surface was wiped clean and cells on the lower surface photographed (top) and then solubilized. Absorbance at 570 nm was measured with a microplate reader. Cell migration is expressed as percentage with respect to RET/PTC3 cells, whose migration was arbitrarily set at 100. Each column represents the average ± SD of 3 independent experiments (bottom). (C) Suppression of endogenous BRAF in PC RET/PTC3 cells was obtained by transfection of RNA interference against BRAF. Cells were counted at different time points, and the average results of 3 independent experiments are reported.