Figure 1. For figure legend, see page 1464.Figure 1 (See previous page).

TGF-β induces changes in DNA methylation during EMT in ovarian cancer cells. (A) Phase contrast microscopy identifies morphological changes induced in SKOV3 cells by TGF-β (5 ng/ml) at 48, 120, and 48 h after removal of TGF-β (magnification 100×, panels; 200×, insets). Western blotting for E-cadherin and vimentin in SKOV3 cells treated with TGF-β for 120 h (right panel). (B) Unsupervised hierarchical clustering displays differential DNA methylation profiles of SKOV3 cells treated with vehicle, TGF-β for 48 and 120 h, or 48 h after removal of TGF-β (n = 3 replicates per group; each group is color coded). Rows represent individual samples and columns represent CpG sites β-values. A visual dual color code is utilized with red and blue indicating high and low expression levels, respectively. The scale of color saturation reflects β-values for individual CpG sites. (C) Unsupervised sample classification based on principal component analysis of DNA methylation profiles of SKOV3 cells treated with TGF-β for the indicated time points (3 replicates per group). Samples with similar profiles cluster together. (D) Average β-values for all CpG sites in SKOV3 cells treated with TGF-β (5 ng/ml) for 48, 120, and 48 h after removal of TGF-ß. (E) Effects of TGF-β on average percent methylation of 4 CpG sites of the LINE-1 sequence at the indicated timepoints were determined by pyrosequencing. Bars represent means of 3 replicates ± SE. * denotes P < 0 .05. (F) Gene networks were ranked by log P-values and compared TGF-β treated versus control SKOV3 cells. Analysis within the top ranked networks (log P-value > 25) displays interconnected genes as nodes. Genes are colored according to expression levels, red symbols corresponding to up-regulated genes and green symbols indicating downregulation. Dashed lines between nodes show indirect interactions; continuous lines indicate direct interactions.