Article Figures & Data
Figures
- Figure 1
Ectopic BORIS expression in ovarian cell lines. (A) Western blot analysis of BORIS and β-galactosidase (LacZ) expression in the indicated cell lines at 48 and 96 hours post-transduction. The approximate position of the molecular weight markers on the BORIS Western blot is shown. α-Tubulin was used as a loading control. L48 and L96; LacZ recombinant adenovirus infection at 48 and 96 hours, respectively. B48 and B96; BORIS recombinant adenovirus infection at 48 and 96 hours, respectively. (B) IF staining of BORIS in OVCAR429 cells 48 hours post-infection. DAPI staining was used as a nuclear staining control. Overlay indicates computational merging of BORIS expression and DAPI staining.
- Figure 2
BORIS expression does not induce CG antigen gene expression in ovarian cell lines. (A) BORIS recombinant adenovirus was used to infect the indicated ovarian cell lines for 48 hours, and RNA was harvested to measure MAGE-A1, NY-ESO-1, XAGE-1, and GAPDH (control) expression by RT-PCR. Cells were treated with 1.0 µM decitabine (DAC) for 96 hours as a positive control for CG antigen gene induction. Sample key is shown at right. (B) LacZ or BORIS recombinant adenovirus was used to infect the indicated ovarian cell lines for 1 or 2 weeks, and RNA was harvested to measure MAGE-A1, NY-ESO-1, XAGE-1, and GAPDH (control) expression by RT-PCR. Cells were treated with 1.0 µM decitabine (DAC) for 96 hours as a positive control for CG antigen gene induction. Sample key is shown at right.
- Figure 3
BORIS overexpression does not alter CG antigen promoter methylation levels in ovarian cell lines. LacZ or BORIS recombinant adenovirus was used to infect the indicated ovarian cell lines for 48 or 96 hours, and genomic DNA was harvested to measure (A) MAGE-A1, (B) NY-ESO-1, and (C) XAGE-1 promoter methylation. The data shown represent the average methylation level of five CpG sites for MAGE-A1, fifteen CpG sites for NY-ESO-1, and ten CpG sites for XAGE-1. As an internal control, pyrosequencing of human testis DNA showed significant hypomethylation of each gene (23.9%, 24.9%, and 21.8% methylation, respectively). (D) LacZ or BORIS was expressed in OVCAR3 and OVCAR429 cells as described above, and genomic DNA was harvested at 1 and 2 weeks post-infection and used to measure NY-ESO-1 promoter methylation by pyrosequencing. The human colorectal cancer cell lines HCT116 and DKO served as internal positive and negative controls for NY-ESO-1 methylation, respectively. Error bars indicate ±1 SD.
- Figure 4
BORIS expression does not alter global DNA methylation in ovarian cell lines. LacZ or BORIS recombinant adenovirus was used to infect the indicated ovarian cell lines, and genomic DNA was harvested at 96 hours post-infection. As a control, cells were treated with 5 µM decitabine (DAC) for 48 hours. (A) 5mdC levels were measured using LC-MS. (B) LINE-1 DNA methylation was measured using bisulfite pyrosequencing. The data shown represent the average methylation level of 3 CpG sites. Error bars indicate ±1 SD.
- Figure 5
Kinetics of CG antigen gene induction and promoter DNA hypomethylation following decitabine treatment. OVCAR429 cells were treated with 1.0 µM decitabine and cell extracts were harvested at the indicated time points post-treatment. (A) qRT-PCR was used to measure CG antigen gene expression. (B) Sodium bisulfite pyrosequencing was used to measure CG antigen gene promoter methylation.
- Figure 6
Effect of BORIS siRNA knockdown on decitabine-mediated CG antigen gene induction in OVCAR3 cells. Two different BORIS-targeting siRNAs, or a control siRNA, were transfected into OVCAR3 cells at a concentration of 50 nM. 24 hours post-transfection, cells were treated with 2.0 µM decitabine (DAC). 48 hours later, RNA extracts were harvested and used for qRT-PCR analysis of (A) BORIS, (B) MAGE-A1, and (C) NY-ESO-1 mRNA expression. Error bars indicate ±1 SEM. Student's t-test P values: *, P < 0.05; **, P < 0.01.
Volume 10, Issue 1
