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. 2018 Oct 5;10(10):371. doi: 10.3390/cancers10100371

Figure 6.

Figure 6

DNA methylation impedes PR binding to progesterone-responsive elements (PREs). (a) Screen shot from the UCSC genome browser showing the CpG island (CpG 89) at the ESR1 promoter and the positions of the canonical PREs containing a CpG (blue line) and six half-palindromic PREs with one or two neighboring CpGs (red lines). (b,c) Electrophoretic-mobility shift assay using the indicated amount of purified human PR to capture the PRE with no CpG (ACAGTTTGT; no CpG), one methylated (MetCpG) or unmethylated CpG (UnmetCpG) (ACGGTTTGT) (b); two methylated (MetCpGs) or two unmethylated CpGs (UnmetCpGs) (ACGGTTCGT) (c). Quantification of the percentage of PR binding to different probes is shown in the lower part of the gel images. Error bars represent the SD of three independent experiments. * p less than or equal to 0.05, ** p less than or equal to 0.01, unpaired two-tailed Student’s t-test. (d) A double-stranded oligonucleotide probe with no CpG (ACAGTTTGT) was incubated with 2.4 μg of purified human PR and analyzed by PAGE either in the absence (–) or presence (+) of 100-fold excess of unlabeled oligonucleotides containing two unmethylated (UNMET) or two methylated (MET) CpGs (ACGGTTCGT). Error bars represent the SD of three independent experiments. *** p less than or equal to 0.005, unpaired two-tailed Student’s t-test. The dashed grey line indicates that a lane between the two samples was removed.