Fig. 4.

Length of BRAF-X1 and BRAF-X2 3’UTR in melanoma. a The analysis of the reads mapping to exon 19 across its entire length indicates that the 3’UTR of BRAF-X1 and BRAF-X2 is as long as 7 kb. A representative example of a primary (left) and a metastatic (right) melanoma case is reported. b Cartoon summarizing the position of the primers and the siRNAs used to determine the length of the 3’UTR of BRAF-X1 and BRAF-X2 in melanoma cell lines. The 4 primer pairs used for real-time PCR amplification of BRAF-X1 plus X2 (BRAF-E19-1/2/3/4 qRT-PCR F/R) are represented as black arrows. The 4 primer pairs used for PCR amplification of BRAF-X1 plus X2 (BRAF-E19-1/2/3/4 F/R) are represented as open pink arrows. BRAF-E19-1 qRT-PCR F and BRAF-E19-1 F have the same sequence. The siRNAs used to knock-down BRAF-X1 plus X2 (si-BRAF-E19-1/2/3) are represented as yellow and black rectangles. The primers used for real-time PCR amplification of all BRAF isoforms (totBRAF qRT-PCR F/R) are represented as red arrows. c PCR performed on A375, 501Mel and MeWo melanoma cells using BRAF-E19-1/2/3/4 primer pairs. Genomic DNA (gDNA) is used as positive control. d Chained PCR performed on A375 melanoma cells using “hybrid” primer pairs: BRAF-E19-1 F with BRAF-E19-2 R, BRAF-E19-2 F with BRAF-E19-3 R, and BRAF-E19-3 F with BRAF-E19-4 R. Genomic DNA (gDNA) is used as positive control. e Northern blot of total RNA extracted from A375, 501Mel, and MeWo cells and hybridized with a labeled probe for BRAF CDS. Considering that the size of BRAF CDS is about 2.3 kb, the band detected in all cell lines is consistent with a 7 kb long 3’UTR. f Expression levels of BRAF CDS (detected using the totBRAF qRT-PCR primers) and of different regions of the 3’UTR transcribed from E19 (detected using the BRAF-E19-1/2/3/4 qRT-PCR primer pairs) after the transfection of the indicated siRNAs. The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM of 3 independent experiments. *p < 0.05