Fig. 4
Ectopic expression of BRAFV600E represses TET1 and TET2 and causes DNA hypermethylation. a Lentiviral BRAFV600E mRNA expression in BRAFV600E (brafV600E) and control (gfp)-transduced Colo320 and Caco2 cell lines normalized to GAPDH and ACTB. Co115 cell constitutively expressing BRAFV600E is used as reference. bTET1 and TET2 mRNA expression (upper) with Western blot analysis of protein levels (lower) in cells from panel a. P values were calculated with Welch two-sample t-test. Error bars denote SD (n = 2). Protein signal quantified by image studio software is relative to gfp. Shown are the representative blot from two independent experiments. c DNA methylation at TET1 and TET2 promoter-associated CGIs by bisulfite-pyrosequencing in cells from panel a. Representation is as in Fig. 2b. d Genome-wide methylation profiles in cells from panel a. Shown are the number of hyper (red) and hypo (green) methylated CpGs. To make analysis comparable between platforms, only CpGs corresponding to HM27K are shown. e Venn diagrams show overlap of hypermethylated CpGs from panel d with CIMP-CpGs identified in colon cancers in Fig. 1. Calculated Fisher's exact test are reported as well as associated odds ratios. f Methylation levels at CIMP markers of a panel previously proposed by Hinoue and coworkers (B3GAT2, KCNK13, RAB31, SLIT1, FAM78A, FSTL1, KCNC1, MYOCD, and SLC6A4) and hMLH1 in Colo320 brafV600E and gfp cells from panel a. Shown are all CpGs present on the array for the corresponding gene; hypermethylated (red), hypomethylated (green) or none (black). g The mRNA expression levels of 12 CIMP-CpG-associated genes in brafV600E cells relative to gfp. P values were calculated with Welch two sample t-test. Error bars denote SD (n = 2). hTET1 and TET2 mRNA expression (upper) in Co115 cells treated with 2 μM PLX4032 (grey) or DMSO (black) for 56 days. At 28 days, cultures were continued with (straight line) or without PLX4032 (dotted line). Shown are expression levels relative to DMSO. Day 0 is shown in open circles. P values were calculated with Welch two sample t-test. Error bars denote SD (n = 5). Western blot for TET1 and TET2 at 14 days in two represented replicates. Protein signal quantified by image studio software is relative to DMSO. Shown are the representative blot from three independent experiments. i Dot blot showing levels of 5hmC at 2 and 14-day timepoints in cells from panel h. Shown are 3 replicates for each timepoint with methylene blue staining (DNA) as loading control. Signal quantified by image studio software is a shown as ratio (PLX4032/DMSO). j DNA demethylation at the TET1 promoter-CGI in cells from panel h. Boxplots show the methylation difference (PLX4032-DMSO) measured by bisulfite-pyrosequencing at 61 CpGs (green circles) with median (line) and mean (red circles). P values were calculated with Wilcoxon rank-sum test