Fig. 6

BRAF transcript variants in the context of acquired resistance to BRAF and MEK inhibitors. a Real-time PCR detection of total BRAF (red), BRAF-ref (grey), BRAF-X1 plus X2 (black), BRAF-X1 (blue), and BRAF-X2 (green) in 451Lu parental cells (P) and in 451Lu-MR resistant cells (MR). The latter show acquired resistance to BRAF and MEK inhibitors due to the focal amplification of the BRAF gene. b Cartoon summarizing the position of the primers and the siRNAs used to determine the presence and the level of the Δ[3–10] variant of BRAF. For details, please refer to Additional file 1: Figure S17. c Real-time PCR detection of total BRAF (red), full length BRAF (brown), Δ[3–10]BRAF (orange, left panel), BRAF-ref (grey), BRAF-X1 plus X2 (black), BRAF-X1 (blue), and BRAF-X2 (green, right panel) in A375 parental cells and in A375 C2 cells. The latter show acquired resistance to vemurafenib due to the presence of Δ[3–10]BRAFV600E splicing variant. d PCR amplification of the reference, X1, and X2 Δ[3–10]BRAF splicing variants from the cDNA of A375 C2 cells. Lane 1: 1 kbp ladder. Lane 2: Δ[3–10]BRAF-ref amplification was obtained using BRAF-E1/2 F primer and refBRAF-STOP R primer (open red and grey arrows in b). Lane 3: Δ[3–10]BRAF-ref CDS was amplified from pMSCVHygro-Δ[3–10]BRAFV600E-ref plasmid and used as positive control. Lane 4: the amplification of Δ[3–10]BRAF-X1 (upper band) and Δ[3–10]BRAF-X2 (lower band) was obtained using BRAF-E1/2 F primer and BRAF-X1-STOP R primer (open red and black arrows in b). Lane 5: Δ[3–10]BRAF-X1 CDS was amplified from pMSCVHygro-Δ[3–10]BRAFV600E-X1 plasmid and used as positive control. Lane 6: Δ[3–10]BRAF-X2 CDS was amplified from pMSCVHygro-Δ[3–10]BRAFV600E-X2 plasmid and used as positive control. e-f Real-time PCR detection of full length BRAF, Δ[3–10]BRAF, BRAF-ref, BRAF-X1 plus X2, BRAF-X1, and BRAF-X2 24 h after the transfection of si-flBRAF (e) and si-Δ[3–10]BRAF (f) in A375 C2 cells. g Real-time PCR detection of full length and Δ[3–10] BRAF 24 h after the transfection of si-refBRAF and si-BRAF-E19-1 in A375 C2 cells. h Western blot of full length and Δ[3–10] BRAFV600E, as well as of pMEK 48 h after the transfection of the indicated siRNAs or siRNA mixes in A375 C2 cells. i Growth curve of A375 C2 cells after the transfection of the indicated siRNAs. Throughout the experiment, the cells were kept in DMSO (left panel) or in 2 uM vemurafenib (right panel). The arrows highlight the increased sensitivity displayed by A375 C2 cells to si-Δ[3–10]BRAF (orange) and si-BRAF-E19-1 (black), when grown in vemurafenib. The graph represents the mean only of 3 independent experiments. (j) Colony formation assay of A375 C2 cells after the transfection of the indicated siRNAs. Throughout the experiment, the cells were kept in DMSO (clean bars) or in 2 uM vemurafenib (dashed bars). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM (or mean ± SD in a and c) of 3 independent experiments. *p < 0.05, **p < 0.01