Nhibitor TSA on the mRNA expression of MAGE-A1 gene and the other family members (MAGE-A2, -A3 and -A12) in different cell lines. Moreover, we assessed the methylation status of the MAGE-A promoters by sodium bisulfite mappingbefore and after stimulation with the demethylating agent 5-aza-CdR and/or TSA. While the methylation patterns clearly correlated with the basal MAGE RNA transcript levels, u
Nhibitor TSA or after transient transfection of these cells with an expression plasmid encoding for BORIS were separated on an agarose gel. The bar chart shows the relative changes in mRNA expression levels of MAGE-A1 in MCF-7 cells by quantitative real-time PCR. The significant p-values are shown (C). H2O lane serves as a negative control. The housekeeping gene -Actin was selected as an internal
Upregulate MAGE-A1 expression in this cell line. BORIS-specific shRNA reduced the BORIS mRNA expression nearly completely in presence and absence of scramble shRNA (Figure 3D, p = 0.0001). Likewise, the downregulation of MAGE-A1 expression by BORIS-specific shRNA was more prominent in MCF-7 cells than in MDA-MB-468 cells. As measured by quantitative real time PCR, BORIS-specific shRNA reduced the
Ontrol. At 48 or 72 hour post-transfection, we quantified the changes in the BORISSchwarzenbach et al. BMC Cancer 2014, 14:796 http://www.biomedcentral.com/1471-2407/14/Page 7 ofAMAGE-A1 429 bp _ ?Actin 202 bp _MCF-BMAGE-A1 429 bp _ ?Actin 202 bp _BCMMAGE-A1 mRNA Expression ( )C80000 60000 40000 20000 2000 1500 1000 500MCF-p=0.p=0.p=0.001 p=0.basalAZATSAAZA + TSABorisFigure 1 Comparison of the MAG
Ontrol. At 48 or 72 hour post-transfection, we quantified the changes in the BORISSchwarzenbach et al. BMC Cancer 2014, 14:796 http://www.biomedcentral.com/1471-2407/14/Page 7 ofAMAGE-A1 429 bp _ ?Actin 202 bp _MCF-BMAGE-A1 429 bp _ ?Actin 202 bp _BCMMAGE-A1 mRNA Expression ( )C80000 60000 40000 20000 2000 1500 1000 500MCF-p=0.p=0.p=0.001 p=0.basalAZATSAAZA + TSABorisFigure 1 Comparison of the MAG
E experiments, we chose, therefore, MCF-7 cells, because they do not express MAGE-A1 mRNA (Table 1). Based on the usually low transfection efficiency we sorted the transfected MCF-7 cells from untransfected cells by FACS and observed a transfection efficiency of about 10 . Subsequently, sodium bisulfite mapping showed a demethylation of the MAGE-A1 promoter of approximately 56 (range from 44 to 6
E experiments, we chose, therefore, MCF-7 cells, because they do not express MAGE-A1 mRNA (Table 1). Based on the usually low transfection efficiency we sorted the transfected MCF-7 cells from untransfected cells by FACS and observed a transfection efficiency of about 10 . Subsequently, sodium bisulfite mapping showed a demethylation of the MAGE-A1 promoter of approximately 56 (range from 44 to 6
Ults show that changes in the BORIS transcript levels are associated with those of MAGE-A1 and corroborate that BORIS is involved in the activation of MAGE-A1 gene expression.BORIS affects the DNA methylation pattern of MAGE-A1 geneFigure 2 BORIS mRNA expression in MDA-MB-468, MCF-7 and BCM1 cells, untreated or treated with 5-aza-CdR. RT-PCR products of BORIS mRNA were separated on an agarose gel.