Figure 3.
SMARCA5 modulates NSD2 localization and H3K36me2 levels in PCH. (A) Candidates of NSD2-interacting proteins that mediate heterochromatic H3K36me2. Among the NSD2-interacting factors previously identified (Huang et al., 2019), some are known to be enriched in PCH. (B) Percentages of cells clearly showing PCH H3K36me2 when the indicated proteins are depleted by shRNA-mediated knockdown. The knockdown efficiencies are indicated on the right as relative expression levels to the control (Scramble) shRNA, measured by RT-qPCR. (C–E) Localization of Halo-SMARCA5 and H3K36me2. iMEFs and Pmi28 cells, transfected with the Halo-SMARCA5 expression vector, were incubated in JF646 HaloTag ligand and Hoechst 33342. After live imaging, cells were fixed and stained with anti-H3K36me2. (C) Single optical sections for Halo-SMARCA5 (green) and Hoechst 33342 (red) in living cells. Contrast was adjusted individually to compare the localization patterns. (D and E) PCH-to-nucleus intensity ratios of Halo-SMARCA5 in live cells (D) and H3K36me2 by immunofluorescence (E). (F–I) Effects of SMARCA5 KO on the localization of H3K36me2 and NSD2. (F) SMARCA5 protein levels evaluated by western blotting in WT and SMARCA5 KO#3 iMEFs. Lamin B served as a loading control. The positions of size standards are indicated on the left. (G and H) Immunofluorescence of H3K36me2 and quantification. (G) WT and SMARCA5 KO#3 without and with Halo-SMARCA5 were stained with anti-H3K36me2 (green) and Hoechst 33342 (red). Single optical section images are shown with individual contrast adjustments to compare the localization patterns. (H) PCH-to-nucleus intensity ratios of H3K36me2. (I) Intensity ratios of Halo-NSD2 in WT and SMARCA5 KO#3 cells without and with SMARCA5-sfGFP or EGFP. See Fig. 1 legend for the details of box plots. Scale bars, 10 μm. Source data are available for this figure: SourceData F3.

SMARCA5 modulates NSD2 localization and H3K36me2 levels in PCH. (A) Candidates of NSD2-interacting proteins that mediate heterochromatic H3K36me2. Among the NSD2-interacting factors previously identified (Huang et al., 2019), some are known to be enriched in PCH. (B) Percentages of cells clearly showing PCH H3K36me2 when the indicated proteins are depleted by shRNA-mediated knockdown. The knockdown efficiencies are indicated on the right as relative expression levels to the control (Scramble) shRNA, measured by RT-qPCR. (C–E) Localization of Halo-SMARCA5 and H3K36me2. iMEFs and Pmi28 cells, transfected with the Halo-SMARCA5 expression vector, were incubated in JF646 HaloTag ligand and Hoechst 33342. After live imaging, cells were fixed and stained with anti-H3K36me2. (C) Single optical sections for Halo-SMARCA5 (green) and Hoechst 33342 (red) in living cells. Contrast was adjusted individually to compare the localization patterns. (D and E) PCH-to-nucleus intensity ratios of Halo-SMARCA5 in live cells (D) and H3K36me2 by immunofluorescence (E). (F–I) Effects of SMARCA5 KO on the localization of H3K36me2 and NSD2. (F) SMARCA5 protein levels evaluated by western blotting in WT and SMARCA5 KO#3 iMEFs. Lamin B served as a loading control. The positions of size standards are indicated on the left. (G and H) Immunofluorescence of H3K36me2 and quantification. (G) WT and SMARCA5 KO#3 without and with Halo-SMARCA5 were stained with anti-H3K36me2 (green) and Hoechst 33342 (red). Single optical section images are shown with individual contrast adjustments to compare the localization patterns. (H) PCH-to-nucleus intensity ratios of H3K36me2. (I) Intensity ratios of Halo-NSD2 in WT and SMARCA5 KO#3 cells without and with SMARCA5-sfGFP or EGFP. See Fig. 1 legend for the details of box plots. Scale bars, 10 μm. Source data are available for this figure: SourceData F3.

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