Figure 1

DNA methylation of the PRKD1 promoter silences PKD1 expression in invasive breast cancer cell lines. (A) Schematic representation of PRKD1 promoter region. CpG sites found by bisulfite sequencing and regions amplified by methylation-specific PCR (MSP-PCR) or in situ MSP-PCR are indicated. (B) PRKD1 promoter methylation was determined for the non- or minimally invasive breast cancer cell lines MCF-7, ZR-75-1 and BT-474; the highly invasive cell lines MDA-MB-231, MDA-MB-468, T47D and BT-20; and the normal MCF-10A cell line. Unmethylated CpG sites are shown as empty squares and methylated CpG sites as filled squares according to their percentage of methylation for all clones analyzed. (C) Percentage of methylation of PRKD1 promoter was determined by bisulfite sequencing. In addition, RNA was isolated from cells, and PRKD1 expression levels were calculated as the sum of the individual exon read counts. (D) Controls for MSP-PCR are shown. Enzymatically methylated or unmethylated DNA was modified by bisulfite treatment, and MSP-PCR was performed using the indicated primers. (E) Genomic DNA from indicated cell lines was modified by bisulfite treatment, and MSP-PCR was performed using methylation-specific primers for the PRKD1 promoter. (F) Indicated cell lines were analyzed for protein kinase D1 (PKD1), PKD2 or PKD3 expression by Western blotting. Immunostaining for β-actin served as a loading control. All experiments were independently performed at least three times.