Figure S2.
Effect of epigenetic modifiers and chromatin remodeling factors on H3K36me2 localization. Single optical sections of confocal microscope images are shown with individual contrast adjustments to compare the localization patterns. (A and B) PCH localization of H3K36me2 in cells lacking H3K9me2/3 (A) or DNA methylation (B). 5KO iMEFs (lacking G9a, Glp, Setdb1, Suv39h1, and Suv39h2) (A) and TKO ESCs (lacking Dnmt1, Dnmt3a, and Dnmt3b) (B) were fixed and stained with anti-H3K36me2 (green) and Hoechst 33342 (red). Single optical sections are shown on the left. Box plots display the intensity ratios of H3K36me2 in PCH to the nucleus. (C) Representative live-cell images of HaloTag-tagged H3K36 methylases and demethylases. NIH3T3 cells transfected with expression vectors for indicated HaloTag-tagged enzymes were stained with JF646 HaloTag ligand (green) and Hoechst 33342 (red). (D) Localization of NSD2 mutant without H3K36me2-binding domain and deficient in catalytic activity. Schematic drawing of NSD2 domain structure and an NSD2 (ΔPWWP1, Y1092A) mutant, which lacks H3K36me2-binding PWW1 domain and is deficient in catalytic activity with Y1092A mutation in SET domain, is indicated on top. Pmi28 cells were transfected with expression vectors of HaloTag alone (Halo), Halo-NSD2 (FL), and Halo-NSD2 (ΔPWWP1, Y1092A) and stained with JF646 HaloTag ligand (green) and Hoechst 33342 (red) for live-cell imaging. Confocal images and PCH-to-nucleus intensity ratios of Halo in live cells are shown on the left and right, respectively. (E–H) Effects of BAZ1A and BAZ2A on H3K36me2 localization. iMEFs and Pmi28 cells transfected with emGFP-BAZ1A (E) or emGFP-BAZ2A (F–H) expression vectors were stained with Hoechst 33342 for live-cell imaging. Cells were fixed and stained with anti-H3K36me2. (E and F) Examples of single optical sections of cells expressing emGFP-BAZ1A (E) or emGFP-BAZ2A (F) (red), H3K36me2 (green), and Hoechst 33342 (blue). (G and H) PCH-to-nucleus intensity ratios of emGFP-BAZ2A in live cells (G) and H3K36me2 by immunofluorescence (H). See Fig. 1 legend for the details of box plots. Scale bars, 10 μm.

Effect of epigenetic modifiers and chromatin remodeling factors on H3K36me2 localization. Single optical sections of confocal microscope images are shown with individual contrast adjustments to compare the localization patterns. (A and B) PCH localization of H3K36me2 in cells lacking H3K9me2/3 (A) or DNA methylation (B). 5KO iMEFs (lacking G9a, Glp, Setdb1, Suv39h1, and Suv39h2) (A) and TKO ESCs (lacking Dnmt1, Dnmt3a, and Dnmt3b) (B) were fixed and stained with anti-H3K36me2 (green) and Hoechst 33342 (red). Single optical sections are shown on the left. Box plots display the intensity ratios of H3K36me2 in PCH to the nucleus. (C) Representative live-cell images of HaloTag-tagged H3K36 methylases and demethylases. NIH3T3 cells transfected with expression vectors for indicated HaloTag-tagged enzymes were stained with JF646 HaloTag ligand (green) and Hoechst 33342 (red). (D) Localization of NSD2 mutant without H3K36me2-binding domain and deficient in catalytic activity. Schematic drawing of NSD2 domain structure and an NSD2 (ΔPWWP1, Y1092A) mutant, which lacks H3K36me2-binding PWW1 domain and is deficient in catalytic activity with Y1092A mutation in SET domain, is indicated on top. Pmi28 cells were transfected with expression vectors of HaloTag alone (Halo), Halo-NSD2 (FL), and Halo-NSD2 (ΔPWWP1, Y1092A) and stained with JF646 HaloTag ligand (green) and Hoechst 33342 (red) for live-cell imaging. Confocal images and PCH-to-nucleus intensity ratios of Halo in live cells are shown on the left and right, respectively. (E–H) Effects of BAZ1A and BAZ2A on H3K36me2 localization. iMEFs and Pmi28 cells transfected with emGFP-BAZ1A (E) or emGFP-BAZ2A (F–H) expression vectors were stained with Hoechst 33342 for live-cell imaging. Cells were fixed and stained with anti-H3K36me2. (E and F) Examples of single optical sections of cells expressing emGFP-BAZ1A (E) or emGFP-BAZ2A (F) (red), H3K36me2 (green), and Hoechst 33342 (blue). (G and H) PCH-to-nucleus intensity ratios of emGFP-BAZ2A in live cells (G) and H3K36me2 by immunofluorescence (H). See Fig. 1 legend for the details of box plots. Scale bars, 10 μm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal