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
L line BCM1, the expression of both genes is opposite: no levels of MAGE-A1 [2^(Ct) 1.07] and high levelsof BORIS [2^(Ct) 24.39]. We transiently transfected expression plasmid encoding BORIS into both cell lines, with negligible transcript levels of MAGE-A1, and quantified endogenous MAGE-A1 mRNA by RT (reverse transcription)-PCR and gel electrophoresis. As depicted in Figure 1, BORIS was able to
L line BCM1, the expression of both genes is opposite: no levels of MAGE-A1 [2^(Ct) 1.07] and high levelsof BORIS [2^(Ct) 24.39]. We transiently transfected expression plasmid encoding BORIS into both cell lines, with negligible transcript levels of MAGE-A1, and quantified endogenous MAGE-A1 mRNA by RT (reverse transcription)-PCR and gel electrophoresis. As depicted in Figure 1, BORIS was able to
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 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
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
Ansfected cells from untransfected cells by FACS analyses.MCF-p=0.p=0.0001 p=0.0001 p=0.Figure 4 Histone signature at the MAGE-A1 promoter as examined by chromatin immunoprecipitation. DNA was derived from unstimulated (basal) MCF-7 cells, 5-aza-CdR- and/or TSA-stimulated MCF-7 cells and MCF-7 cells transfected with the expression plasmid encoding for BORIS. DNA-bound histones were immunoprecipita
Ansfected cells from untransfected cells by FACS analyses.MCF-p=0.p=0.0001 p=0.0001 p=0.Figure 4 Histone signature at the MAGE-A1 promoter as examined by chromatin immunoprecipitation. DNA was derived from unstimulated (basal) MCF-7 cells, 5-aza-CdR- and/or TSA-stimulated MCF-7 cells and MCF-7 cells transfected with the expression plasmid encoding for BORIS. DNA-bound histones were immunoprecipita