Effect of IFN-β treatment on xaf1 transcript and protein levels in selected cell lines. (A) Schematic representation of the 8 CpG sites of the 5' upstream region of the xaf1 gene. (B) Methylation status of xaf1 promoter region in selected cell lines was determined based on analysis of 8 CpG sites by methylation-sensitive PCR. Genomic DNA was treated with sodium bisulphite. PCR products with MS2 (P1) and MS3 (P2) primers were cloned into the TopoTA vector and sequenced. Ten cloned PCR products were sequenced for each cell line. Black, dark gray/light gray and open circles represent complete methylation, partial methylation and unmethylation at each CpG site, respectively. Controls are total blood DNA from healthy volunteers. (C) Graph representing the percentage of average methylation at all sites in selected cell lines. (D) xaf1 transcript levels were assessed by quantitative RT-PCR on total RNA and indexed to GAPDH transcript levels in non-treated cells (NT) and cells exposed to 500 U/mL of IFN-β for 24 hours. The protein level of endogenous (E) or induced (F) XAF1 was determined by western blot analysis, 30 μg of total protein extract were resolved by 12% SDS-PAGE and XAF1 was detected using an anti-XAF1 mouse monoclonal antibody. Membranes were stripped and re-probed with anti-β-actin antibody as a loading control. (F) Cells were left untreated (NT), exposed to 500 U/mL of IFN-β (IFN) for 24 hours or treated with 5-AZA-dC at the indicated concentration, followed 24 hours later by IFN-β for 24 hours. The blot in (E) was intentionally overexposed to increase the weak signal of XAF1 in SF295 cells (F).