CXCL12 and ESR1 methylation analyses of breast normal and tumour cell lines and MSP in primary breast tumours. (A) MSP was performed using bisulphite treated DNA with primers that recognize methylated promoter only. The bands represent positive results for the methylation in the islands 2 and 4 for CXCL12 and ESR1 genes. H2O was used as template in the negative control (NC). (B) Nine of sixty-nine representative samples are included in the figure. Tumour samples were subjected to the MSP reaction, confirming the standardization done with the cell lines for the methylated condition. The results for ESR1 showed promoter methylation in the samples 1 to 7 and 9. The tumour 1 presented both islands negative for methylation of CXCL12 gene; however ESR1 methylation was present even thought this sample is ERα positive. The tumour 8 represented a sample with unmethylated ESR1. The lanes 1, 3 and 8 were representative sequenced samples after bisulphite treatment (see Figure 1D).