OVOL1 and OVOL2 induce MET in mesenchymal prostate cancer cells.

<div><p>(A) qPCR: mRNA expression in PC3-EMT14-sh4 relative to PC3-EMT14-Scr.</p> <p>(B) Bright-phase microscopy: PC3-EMT14 cells expressing OVOL1 and OVOL2 exhibit an epithelial phenotype as compared to the parental PC3-EMT14. Scale bars are 100 µm.</p> <p>(C) qPCR: Expression of the epithelial cell markers E-cad and ESRP1, and the EMT-inducing TFs ZEB1, ZEB2, Slug, Snail, and Twist1 in PC3-EMT14 cells expressing OVOL1 and OVOL2 relative to the empty-vector (EV) control.</p> <p>(D) Immunoblot: OVOL1 and OVOL2 protein expression in transfected cells was confirmed by using OVOL1 or V5-specific antibodies, respectively.</p> <p>(E) Immunoblot: Expression of epithelial marker E-cad and mesenchymal markers ZEB1 and Vimentin. Changes in Slug expression were minimal and did not correlate with MET. PC3-EMT14 cells expressing IRF6 or ANKRD22, EV and epithelial PC3-Epi are controls.</p> <p>(F) Invasion/migration assay: Representative images and graphs of cancer cell invasion using a Boyden chamber assay. Bar graphs of OVOL1 and OVOL2 expressing PC3-EMT14 cells relative EV. Percent invasion represents the ratio invading/migrating cells. The graph is representative of one out of three independent experiments.</p> <p>(G) qPCR: Expression of PC3-Epi cells transfected with OVOL1-shRNAs (a, b, c) or OVOL2-shRNAs (d, e, f), relative to the non-silencing control PC3-Epi-NS (represented by the dashed line).</p> <p>(H) Immunoblot: PC3-Epi cells transfected with both OVOL1 and OVOL2 shRNAs (sh-OVOL1/2) demonstrate a decrease in E-cad and increase in Vimentin, which suggests a partial EMT transformation.</p> <p>(I) Schematic: The ZEB1 promoter with potential OVOL2 binding sites (orange triangles) according to the general consensus: 5’-A(A/T) (A/T) (C/A) (T/C)GTTA(T/A). Designed TaqMan primer-pairs are shown as black arrows. Numeration is relative to the transcriptional start site (+1). The sequence of a putative OVOL2 binding site that corresponds to the ChIP DNA in the OVOL2 expressing cells is at +320.</p> <p>(J) ChIP qPCR: (Left) shows input chromatin of PC3-EMT14-OVOL2 relative to EV, and demonstrates that similar amounts of DNA were used (Right). depicts the ChIP DNA using OVOL2 antibody. Primers are named after their forward primer (see panel I). Results were normalized to input controls. The graph depicts one representative experiment out of three with similar results.</p> <p>qPCR results were normalized to β –actin. Graphs show mean +/- sem; p-values are represented as * p < 0.05; ** p < 0.01; *** p < 0.001. The qPCRs and immunoblots are representative of two independent experiments with similar results. See also Figure S2.</p>