Validation of methylated targets in LNcaP and DU145 cells. A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of < 0.05 comparing parental to non-invasive cells and ** comparing parental to invasive cells. C) Staining of invasive or non-invasive cells was performed directly on the Matrigel membrane. Cells were incubated with either anti-pBMX antibody or SOX1 overnight and goat anti-rabbit Alexa-488 was added for 1 hour. Membranes were mounted on glass slides with Vectashield containing DAPI and visualized with a Zeiss-510 L5 confocal microscope. Images were analyzed using the Zeiss LSM5 Image Browser (20×) and further prepared in Adobe Photoshop CS. Increased levels of pBMX and SOX1 are seen in invasive cells compared to the non-invasive cells on top of the membrane.