Fig 2: Chromosomal rearrangement at 7q32 and 7q34 results in a highly expressed SND1-BRAF fusion protein.(A) Western Immunoblot of total protein lysates identify a higher molecular weight band (arrowhead) recognized only by the anti-C-terminal BRAF antibody, and present exclusively in GTL16R1 and GTL16R3, consistent with a fusion event within BRAF. Additionally, the putative fusion BRAF is highly expressed and hyperphosphorylated compared to wild-type BRAF (arrow in BRAF panels). (B) 5' RACE identified the nucleotide sequence of fusion junction spanning exon 16 of SND1 and exon 9 of BRAF. (C) Schematic representation of CNV amplification of the N-terminal portion of SND1 (mapping from exon 1 to 16) and the C-terminal protein of BRAF (mapping from exon 9 to exon 18).

Fig 3: Copy Number Variation (CNV) indicates that BRAF is amplified and bisected in resistant clones.Relative to GTL16, CNV analysis identifies a region of increased copy number at 7q34, with a breakpoint within the BRAF locus in GTL16R1 and GTL16R3. In conjunction with Affymetrix expression analysis, genes within the amplified regions are coordinately over-expressed (indicated in red) normalized to GTL16. Expression of the BRAF transcript is increased over GTL16.

Fig 4: Effect of lactate on oncogene-induced senescence in human lung epithelial cells. a K-Ras activity was analyzed by using Ras Assay Kit according to the manufacturer’s instructions at day 3 after infection with K-Ras (G12S) and vector. Ras represents total Ras, Ras-GTP represents active Ras. b SA-ß-gal activity were analyzed at day 3 after infection of B2B cells with K-Ras (G12S) construct or vector with indicated lactate treatment. The percentage of SA-ß-gal positive cells is presented in the right histogram. The bars represent the mean ± s.d. of triplicates. *P < 0.05, **P < 0.01, ***P < 0.001. c ?H2AX positive cells was analyzed with fluorescence microscopy at day 3 after infection of B2B cells with K-Ras (G12S) construct and then treatment with lactate (0, 10, 20 mM) for 3 h. d Cell cycle analysis was performed at day 3 after infection of K-Ras (G12S) and then treatment with lactate (0, 10, 20 mM) for 3 h. The percentage of G1/S and G2/M phase cells was quantified based on the content of DNA and the data are demonstrated as shown. e Representative Western blot analysis of Snail, p16INK4a, p21CIP-1 and Ras in B2B cells transfected with K-Ras (G12S) and vector at 72 h after transfection and different concentrations treatment of Lactate for 3 h. f Transfection of B2B cells with PTEN (C124S) and B-RAF (V600E) for 72 h then treated with different concentrations Lactate for 3 h were analyzed by immunoblotting of the expression levels of Snail, p16INK4a, and p21CIP-1

Fig 5: miR-193a-3p functions as a tumor-suppressor in colorectal cancer cells and its expression was decreased by overexpression of a BRAF protein. a Transfection of precursors of miR-193a-3p increased the expression of mature miR-193a-3p (top), and inhibited cell viability of the three cell lines irrespective of their KRAS/BRAF mutational status (bottom). b, c Transfection of the precursors of miR-193a-3p inhibited cell invasion ability in RKO and HCT116 cells. d miR-193a-3p overexpression reduced the mRNA levels of EMT-related genes ZEB1, SNAI1, and SNAI2 in RKO cells. e The mutant BRAF (V600E) overexpression activates its downstream pathway. Western blot analysis shows that the overexpression of mutant BRAF protein caused an increase in phosphorylated levels of BRAF and its downstream MEK and ERK, in the HCT8, LIM2405, SW48 and DiFi colorectal cancer cells compared to those transfected with a control vector. f miR-193a-3p expression was decreased in the KRAS/BRAF-wild-type SW48 and DiFi cells, but not in the KRAS-mutant HCT8 cells and the BRAF-mutant LIM2405 cells 72 h after overexpression of mutant BRAF protein. The Student’s t test was used to analyze statistical differences