Fig. 5

BRAF isoforms together account for BRAF functions in melanoma cells. a Cartoon summarizing the position of the primers and the siRNAs used to determine the contribution of reference, X1 and X2 isoforms to BRAF activities in melanoma cell lines. The primers used for real-time PCR amplification of all BRAF isoforms (totBRAF qRT-PCR F/R), BRAF-ref (refBRAF qRT-PCR F/R), and BRAF-X1 plus X2 (BRAF-E19-1 qRT-PCR F/R) are represented as red, grey and black arrows, respectively. The siRNAs used for the knock-down of the different BRAF isoforms are schematically represented as rectangles: yellow and red, for the knock-down of all BRAF isoforms (si-totBRAF); yellow and grey, for the knock-down of BRAF-ref (si-refBRAF); and yellow and black for the knock-down of BRAF-X1 plus X2 (si-BRAF-E19-1). b Real-time PCR detection of total BRAF, BRAF-ref, and BRAF-X1 plus X2 24 h after the transfection of the indicated siRNAs in A375 cells. c Western blot of BRAF and of its substrate pMEK 48 h after the transfection of the indicated siRNAs in A375 cells. d Growth curve of A375 cells after the transfection of the indicated siRNAs. e Wound healing assay performed using A375 cells transfected with the ndicated siRNAs. The pictures were taken 24 and 36 h after the removal of the silicone inserts. f Xenograft in zebrafish embryos of A375 cells stably expressing mCherry and transfected with the indicated siRNAs. The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. Hpf: hours post fertilization. Scale bar: 100 um. The graphs represent the mean ± SEM of 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001