Fig 2: (A) Heat map hierarchical cluster analysis of CpG probesets in RASAL2 genome identified in a panel of prostate tissues (used in genome-wide DNA methylation analysis [33]). African American (AA) benign tissues (Nl) and AA cancer tissues (CA); European American (CAU)-Nl and CA. (B) Ingenuity Pathway Analysis of TNFα and RASAL2 crosstalk show a crosstalk between TNFα and RASAL2 (p value < 0.05). (C) Expression of RASAL2 and GAPDH by PCR analysis. Negative controls: water, no template, mouse 3T3 cells. Benign prostate cell lines: PNT1A and RWPE1. Prostate cancer cells: LNCaP, PC3 and DU145. Kidney cancer cell line; E006AA.

Fig 3: (A) siRNA-mediated knockdown transfections. PC3 cells were transiently transfected with one of three different RASAL2-specific siRNA or in combinations, or with a scramble nonsense siRNA (negative control). At the indicated times after transfection, cells were trypsinized and counted using a Coulter counter. Data shown are representative of 3 independent experiments. (B) Protein extracts were collected from PC3 cells at the day 3 after transfection with the siRNAs and analyzed by Western blotting with the following antibodies: RASAL2-Myc tagged antibody, C-myc, NF-kB, N-RAS, TNFα, AR, PTEN and GAPDH. Significant difference in cells transfected with RASAL2-specific siRNAs compared to negative control (scrambled siRNA; -ve control) using Fisher t-test (statistical significance is shown as * P < 0.05). Data shown are representative of 3 independent experiments.

Fig 4: Transient transfection of RASAL2 in LNCaP and PC3 prostate cancer cells.(A) The LNCaP PCa cell line was transfected with a RASAL2 cDNA cloned in the mammalian expression vector (pCMV-Myc-DDK) or the pCMV vector only. At the indicated times after transfection, cells were trypsinized and counted using a Coulter counter. Similar transfections were done in PC3 PCa cell line (B). (C) Protein extracts were collected from LNCaP cells at day 3 after transfection with RASAL2 expression plasmid or empty vector and analyzed by Western blotting with the following antibodies; RASAL2-Myc tagged antibody, C-myc, NF-kB, N-RAS, TNFα, AR, PTEN and GAPDH. Densitometry of western blot analysis is shown as the ratio of each protein expression to GAPDH protein. Similar experiments were carried out in PC3 cells (D). Statistical significance is indicated as (* p < 0.05; t-test). Data shown are representative of 3 independent experiments.

Fig 5: (A) Cell growth and morphology of stably transfected PC3 with RASAL2 vector or empty vector compared to untransfected cells. (B) Wounding assay of scatter/migration. PC3 cells stably transfected with RASAL2 plasmid or vector only were used in a scratch wound assay as described in Materials and Methods. The cells were permitted to migrate to the area of clearing for a total of 48 hrs and photomicrographs taken at 0, 24 and 48 hrs. Results shown are typical of 3 separate experiments. (C) qRT-PCR of RASAL2 expression in stably transfected cells (D) Western blot analysis of PC3 cells stably transfected with RASAL2 vector or empty vector only. Densitometry of western blot analysis is shown as the ratio of each protein expression to GAPDH protein. Statistical significance is indicated as *(p < 0.05; t-test). Data shown are representative of 3 independent experiments.