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. 2010 May 10;107(21):9671–9676. doi: 10.1073/pnas.1000401107

Fig. 1.

Fig. 1.

KDM8 H3K36me2 demethylation in MCF7 breast cancer cells. (A) KDM8 and H321A, an inactive enzymatic mutant, were induced to express as Flag fusion proteins. Indirect immunofluorescence with antibodies against Flag (red staining) and methylated H3K36me1, H3K36me2, or H3K36me3 (green staining) was used to analyze in vivo substrate specificity of KDM8. DAPI staining (blue) indicates nuclei location in each field. Cells overexpressing KDM8 (arrows) showed significant loss of H3K36me2 staining, which was dependent on the active JmjC domain, and not observed in the H321A overexpressed cells. (B) Quantitative analysis of Dox-induced KDM8/H321A MCF7 H3K36me2 stained cells from 10 random fields. Eighty percent of induced KDM8 cells showed diminished H3K36me2, whereas 60% of H321A-induced cells exhibited increases in H3K36me2 staining. Percentage of cells that exhibited no change in H3K36me2 is not shown. (C) Histones extracted from Dox-induced KDM8/H321A MCF7 were analyzed by Western blotting with antibodies against H3K36me2 and H3K9me3. A decreased signal in H3K36me2 was observed in KDM8 overexpressed cells (lane 2 vs. lane 1), which was dependent on the active JmjC domain (inactive mutant lanes 3–4). (D) MS analysis of observed in vitro KDM8 demethylase activity. Reactions using H3K36me2 peptide combined with either GST KDM8 101-C WT or GST-KDM8 101-C H321A show 14 Da shift in WT reactions only (asterisk).