Figure 3

Pharmacologic inhibition of PRKD1 methylation leads to PKD1-dependent reversion of the invasive phenotype. In all experiments, cells were treated with 10 μM decitabine or dimethyl sulfoxide (DMSO) as control for 3 days. (A) DNA from MDA-MB-231 treated cells was modified by bisulfite treatment, and methylation-specific PCR was performed with specific primers for the methylated PRKD1 promoter. (B) RNA was isolated from treated cells, and RT-PCR using specific primers for protein kinase D1 (PKD1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression was performed. (C) Lysates from treated cells were analyzed by Western blotting for expression of PKD1, PKD2, PKD3 or β-actin as a loading control. Values represent densitometry calculations (values obtained for MCF-7 cells were set to 1). Intensities of bands were calculated using Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to bands obtained with β-actin. (D) MDA-MB-231 control and PKD1 short hairpin RNA (shRNA) cells (shRNA sequence 2) were treated with 10 μM decitabine or DMSO as a control. Cell viability was determined using a tetrazolium dye assay. The results presented are the means ± SD. (E) MDA-MB-231 control and PKD1 shRNA cells (shown are two different shRNA sequences, 1 and 2) were treated with 10 μM decitabine or DMSO as a control and seeded onto Matrigel-coated transwell filters. Transwell invasion assays were performed over a period of 16 h. scr-shRNA, scrambled control short hairpin RNA. The results presented are means ± SD. P values were calculated using a two-tailed Student’s t-test and standard deviations. For all analyses, P < 0.05 was considered significant. All experiments were independently performed at least three times.