NSD2 is the enzyme responsible for H3K36me2 at PCH. (A–D) Effect of Halo-NSD2 expression on H3K36me2 localization. iMEFs and Pmi28 cells were transfected with the Halo-NSD2 expression vector and treated with JF646 HaloTag ligand and Hoechst 33342. After live imaging, cells were fixed and stained with anti-H3K36me2. (A and B) Examples of single optical sections for Halo-NSD2 (red), H3K36me2 (green), and Hoechst 33342 (blue). Halo-NSD2 expressed (NSD2+) and unexpressed (NSD2−) iMEFs (A) and Pmi28 cells (B) are shown. Contrast was adjusted individually to compare the localization patterns. (C and D) PCH-to-nucleus intensity ratios for Halo-NSD2 in live cells (C) and H3K36me2 by immunofluorescence (D). (E–G) Effect of NSD2 KO on H3K36me2 localization. (E) The protein levels of NSD2 evaluated by western blotting in WT and NSD2 KO#1 and KO#2 iMEFs. Lamin B served as a loading control. Positions of size standards are indicated on the left. (F and G) Immunofluorescence and quantification. (F) WT, NSD2 KO#2, and KO#1 without and with Halo-NSD2 were stained with anti-H3K36me2 (green) and Hoechst 33342 (red). Single optical section images are displayed with individual contrast adjustments to compare the localization patterns. (G) PCH-to-nucleus intensity ratios for H3K36me2. See Fig. 1 legend for the details of box plots. Scale bars, 10 μm. Source data are available for this figure: SourceData F2.