Fig. 3

MDB2 mediates silencing of the HTRA1 promoter. a Fold expression of MBD2 and HTRA1 in HCT116 cells stably downregulated by different shRNAs (1 and 2) or random RNA sequence (nonsense RNA) compared to the vector control (set to 1). Standard deviation is indicated (n ≥ 2 independent experiments done in triplicates, two-tailed Mann–Whitney U test, p-values HTRA1 expression: shMBD2.1 = 0.0039, shMBD2.2 = 0.0028, Nonsense RNA = 0.0028; p-values MBD2 expression: shMBD2.1 = 0.0002, shMBD2.2 = 0.0002, Nonsense RNA = 0.0034)). b ChIP analyses of MBD2 binding the human HTRA1 promoter (−453 bp to −336 bp) in HCT116 and SW480 cells. Results are expressed as fold enrichment compared to the reference GAPDH promoter (n = 4, 2 independent chromatin fractions, unpaired two-tailed t-test, p-value <0.001) (left). ChIP analyses of H3K9 acetylation at the human HTRA1 promoter (−453 bp to −336 bp). Results are expressed as fold enrichment compared to the reference histone 3 (n = 3, 2 independent chromatin fractions, unpaired two-tailed t-test, p-value <0.001) (right). c Electric mobility shift assay using methylated or unmethylated 12 bp double stranded oligonucleotide from the CpG island of the HTRA1 promoter and purified MBD2b in equimolar concentrations (70 pmol). MBD2-DNA complex formation was analysed on 12 % agarose gels. d Proteolysis of MBD2b by trypsin. 5 μg of purified MBD2b were incubated with the amounts of trypsin indicated for 20 min at 37 °C. Samples were analysed on SDS PAGE (upper panel). Proteolysis of MBD2b with bound methylated oligonucleotide by trypsin. The protected MBD2b fragment is boxed (lower panel)