Fig. 7. DNMT1 depletion inhibits Aur-B-mediated histone H3 phosphorylation at pericentromeres. (A) Transfection with antisense oligonucleotide (AS) resulted in >90% depletion of DNMT1 compared with a mismatched oligonucleotide control (MM). Vimentin was detected as a loading control in western blots. (B) Reduction in Aur-B and phospho-H3 is not greater than 50% in these populations. (C-N) Microscopic analysis of Aur-B, phospho-H3 and HP1 in cells transfected with DNMT1-AS and DNMT1-MM oligonucleotides. (C-G) Aur-B and centromere colocalization in cells transfected with DNMT-MM (C,D) and DNMT1-AS (E-G). Examples of cells without (C,E labelled -) and with (D,G labelled +) focal accumulations of Aur-B are shown, as was a third population seen in DNMT1-AS-treated cells that showed a diffuse localization of Aur-B (F, labelled +/-). (H) Histogram summarizing quantification of Aur-B and H3S10P pericentromeric foci in oligonucleotides-treated cells. In each case the DNMT1-MM population was used as a standard, revealing a 62% and 38% decrease in DNMT1-AS cells exhibiting Aur-B and phospho-H3 pericentromeric foci, respectively. (I-N) HP1 and centromere colocalization in cells transfected with DNMT1-MM (I,J) and DNMT1-AS (K,L). Examples of cells exhibiting no focal accumulations of HP1 (I,K labelled -) and with focal accumulations (J,L labelled +). Bars, 10 µm. (M) Histogram represents quantification of cells with focal accumulations of HP1 with DNMT1-MM (blue), set as the standard in comparison with DNMT1-AS (red). Number of analyzed nuclei is reported above. (N) Histogram of percentage of mitotic cells scored in the DNMT1-MM (blue) and the DNMT1-AS (red) populations. Number of analyzed nuclei is reported above.