Figure S1 - Misbehaviour of XIST RNA in Breast Cancer Cells

Effects of BRCA1 RNAi on XIST expression and promoter methylation status in breast cancer cell lines. A) Effects of BRCA1 RNAi on XIST expression in MDA MB 231(XCI type 2) breast cancer cell line. Cells transfected with a mix of two BRCA1-specific siRNAs, mapping to exons 12 and 24, or a control siRNA. After a 72 hrs, cells were processed for BRCA1 immunofluorescence or RNA purification. BRCA1 is immunostained in green and nuclei are marked with DAPI. The histogram represents quantitative RT-PCR analysis performed on cDNA before and after BRCA1 silencing, using primers specific for spliced and unspliced XIST RNA. XIST levels are expressed as a ratio to GAPDH mRNA levels after subtraction of background signal from cDNA synthesis reactions lacking reverse transcriptase. To facilitate comparison between cell lines with different XCI status, the ratio of XIST��GAPDH transcripts was normalised relative to normal HMEC. Error bars represent standard deviation. B) Assessment of XIST promoter methylation levels, by pyrosequencing, in HMEC and MCF7. The percentages of methylation at four CpG positions before and after BRCA1 silencing are reported. In brackets the mean methylation is indicated. The MCF7 promoter shows demethylation after BRCA1 silencing. (5.88 MB EPS)