Fig. 2
DNA methylation causally underlies EMT and resistance to therapy. a Methylation of EMT marker gene promoters. Shown are means ± SEM of the differences in DNA methylation (Δß values) between resistant and parental cells, of all CpGs profiled in the promoters of epithelial (117; gray) and mesenchymal (275; red) marker genes. Significance of the difference between mesenchymal and epithelial marker promoter methylation changes was calculated using a one-sided Mann-Whitney U test. b Correlation of EMT marker expression changes with promoter methylation changes. Shown are, per EMT marker gene, promoter methylation differences (difference in the average of CpGs, x-axis) versus differences in their expression (log2 fold-change, y-axis), between resistant and parental cells. Upon EMT, mesenchymal marker genes (red dots) are expected in the upper left quadrant and epithelial (gray dots) in the lower right quadrant. c EMT expression profile after pharmacological DNA demethylation using 5-aza-2′-deoxycytidine (0.5 μM, 8 days). Shown are differences in expression between resistant and parental cells of 16 epithelial (gray) and 34 mesenchymal (red) marker genes, as determined using RT-qPCR. Significance of the difference between epithelial and mesenchymal gene expression changes was calculated using a two-sided Mann-Whitney U test. d Growth of parental (black) and resistant (red) demethylated cells following exposure to the associated drugs at the parental cells’ estimated IC50 (Table 1), after an 8-day treatment with 0.5 μM 5-aza-2′-deoxycytidine, relative to control cells that were similarly demethylated but not exposed to the drugs. Shown are individual data points and means of experiments performed in triplicate (except for HCC4006P1 and HCC4006ER, HCC827P and HCC827GR, n = 4 and IGROV-1 and IGROV-1/CDDP, n = 2), each replication consisting of nine technical replicates. Significance of the difference between parental and resistant cell growth was calculated using a one-sided Mann-Whitney U test. ns non-significant, *p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001