Figure 5.

Treatment of PC-3 Zeb1^KD prostate cancer cells with the global demethylating agent 5-azacitadine (5-aza) results in decreased DNA methylation, migration and invasion. (A,B) PC-3 Zeb1^KD (Zeb1 knockdown) with doxycycline (Dox) or control (ctrl) cells were treated with either dimethyl sulfoxide (DMSO) or 5-aza (5 µM) for 24 h and DNA was extracted to assess for global DNA methylation via dot blot assays. Representative dot blots are shown. Methylated and unmethylated DNA controls were used to validate 5-methylcytosine (5mC) expression. (C,D) Cells were seeded onto physical barrier cell culture dishes and grown to 90–100% confluency. Treatments (5 µM 5-aza or DMSO) were added to cells, the physical barrier was removed, and cells were allowed to migrate into the wound. (E,F) Cells were seeded onto ultra-low attachment plates and allowed to grow for 96 h to create spheroids. After 96 h of growth, Matrigel and 5 µM 5-aza or DMSO were added. Representative images are shown for each assay; with migration or invasion calculated based on 5 high-powered fields of view (HP-FOV) per well. Scale bars = 300 µm. Data is presented as the mean ± standard error of the mean (SEM) (n = 3). η = significantly different than ctrl with Dox and treated with DMSO. θ = significantly different than Zeb1^KD with Dox and treated with DMSO. ι = significantly different than ctrl with Dox treated with 5 µM 5-aza. κ = significantly different than Zeb1^KD with Dox treated with 5 µM 5-aza (p ≤ 0.05).