Figure 1

Isolation and MeDIP analysis of invasive prostate cancer cells. Matrigel-coated 24-well inserts (8 μM pore size) and non-coated control inserts purchased from BD Biosciences were used according to manufacturer's instructions. A range of 70,000-100,000 cells was seeded for the invasion. Cells were seeded in serum-free RPMI and migrated toward media specific for stem cells (termed SCM). For the isolation of DNA from top "non-invading" and bottom "invading" cells, parallel invasion chambers were set up. For non-invading cells, the bottom of the membrane was scrubbed with a cotton swab, and cells on top were harvested using 500 μL of trypsin incubated at 37°C for 5 minutes. To obtain the invading cells, the top of the membrane was scrubbed with a cotton swab, and the membranes were placed at -80°C until DNA extraction was performed or the cells were harvested with trypsin, as previously mentioned. DNA was extracted using a DNeasy kit (Qiagen), where the cell-based protocol was used to isolate DNA for the top cells, while the tissue extraction method was used to isolate the invaded cells from the previously stored membrane. Total genomic DNA was digested with MseI overnight, and methylated DNA was then collected using MethylCollector kit (Active Motif). Input and methylated DNA was then amplified with 2 rounds of PCR and labeled with either Cy5 for methylated or Cy3 for total DNA. The samples were combined and applied to Agilent's Human Promoter Tiling Array for 40 hours at 65°C. The arrays were then scanned with an Axon scanner (4000B) using GenePixPro version 6.1. The data were extracted using Agilent's Feature Extraction software 9.3.5.1, and differences in promoter methylation of genes in non-invasive and invasive cells were compared using Agilent's ChIP Analytics software version 4.0. Selected targets were then verified for methylation status using methylation-specific PCR, qRT-PCR and ICC.