Additional file 1: of The landscape of BRAF transcript and protein variants in human cancer

Figure S1. Schematic representation of the exons belonging to the different variants of human BRAF reported in NCBI and Ensembl. Figure S2. Scan of exon 18 of BRAF in 8 cancer types. Figure S3. Count of the reads mapping to all BRAF exons, E19 included. Figure S4. Count of reads mapping to all BRAF exons (w/o E19 on the left and w E19 on the right). Figure S5. Count of E13-E16 exon-spanning reads. Figure S6. BRAF-004 transcript variant is expressed but it is truncated. Figure S7. Position of the primers/siRNAs used to detect/downregulate BRAF-ref, BRAF-X1, and BRAF-X2. Figure S8. Real-time PCR detection of BRAF-ref, BRAF-X1 plus X2, BRAF-X1, and BRAF-X2. Figure S9. Box plot of the reads that span E17-E18.2, E18.2-E18b, E18.2-E19, and E17-E19. Figure S10. Stability of reference and X1/X2 BRAF mRNA. Figure S11. Correlation among the expression levels of the different BRAF isoforms in breast cancer, head and neck cancer, lung SCC, AML, and DLBCL. Figure S12. Lack of association of reference, X1, and X2 BRAF levels with age (left panels), gender (middle panels), and stage at diagnosis in primary and metastatic melanoma patients. Figure S13. Lack of association of age (left panels), gender (middle panels), and stage at diagnosis (right panels) with the ratio between BRAF-X1 and BRAF-ref levels (upper panels, red) and with the ratio between BRAF-X2 and BRAF-ref levels (lower panels, blue) in primary and metastatic melanoma patients. Figure S14. Tools for the detection of BRAF CDS plus the E19-derived 3’UTR. Figure S15. Length of BRAF-X1 and BRAF-X2 3’UTR in melanoma. Figure S16. siRNA-mediated downregulation of BRAF isoforms in melanoma cells. Figure S17. Cartoon summarizing the position of the primers and the siRNAs used to determine the levels and the identity of the Δ[3–10] splicing variant of BRAF. Figure S18. Real-time PCR detection of BRAF-ref (grey) and BRAF-X1 plus X2 (black) in vemurafenib-resistant clones and clonal populations. Figure S19. The 3’UTR of X1 and X2 Δ[3–10]BRAF splicing variant is up to 7 kb long. Figure S20. Sequence of reference, X1, and X2 BRAF proteins. Figure S21. Alignment between the sequence of human BRAF-ref (NP_004324.2, left) or BRAF-X1 (XP_005250102.1, right) and mouse Braf-ref (NP_647455.3). Figure S22. Alignment between the sequence of human BRAF-ref (NP_004324.2, left) or BRAF-X1 (XP_005250102.1, right) and rat Braf-X1 (XP_001070228.2). Figure S23. Alignment between the sequence of human BRAF-ref (NP_004324.2, left) or BRAF-X1 (XP_005250102.1, right) and pig Braf-X1 (XP_005654324.1). Figure S24. Dot plot of the secondary structure of BRAF-X1 (blue) and BRAF-X2 (green) mRNA sequences. Figure S25. Enlargement of box1 as reported in Figure S24. Figure S26. BRAF peptides identified by an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, a) and by a 5600 TripleTOF mass spectrometer (ABSciex, b) in A375 melanoma cells. Figure S27. BRAF peptides identified by LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) in WM793B melanoma cells. Figure S28. Translation efficiency of reference, X1 and X2 BRAF mRNAs in A375 cells. Figure S29. BRAF-X2 protein displays a faster decay due to increased proteosomal-mediated degradation. (PDF 14500 kb