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. 2026 Feb 5;54(4):gkag089. doi: 10.1093/nar/gkag089

Figure 5.

Figure 5.

Acute treatment with GSK significantly decreases DNMT1 mobility. (A) Decreased CpG methylation levels in cells treated with the DNMT1-specific inhibitor GSK-3484862 (GSK). Cells were treated with 4 μM GSK for 4 or 24 h, then gDNA was extracted from these samples and subjected to nanopore sequencing. The % methylation difference between the DMSO- and GSK-treated cells is shown. (B) Cells were treated with DMSO or 4 μM GSK for 4, 24, or 48 h, harvested using Laemmli buffer and proteins separated on a 3–8% Tris-Acetate gel. DNMT1-Halo tagged protein was visualized by incubating cells with 500 nM JF646 Halo-ligand for 30 min before harvesting. Coomassie staining of ∼ 205 kDa region is shown as a loading control. (C-F) Cells were treated with either DMSO or 4 μM GSK for 4 h. Images were acquired using HiLo microscopy at ∼ 97 fps over a 10 s period with no delay. Spot-on analysis: (C) Pie chart distribution of the DNMT1 molecules following either DMSO or GSK treatment for 4 h. (D-F) Effect of 4 h GSK treatment on DNMT1 diffusion and chromatin binding. Violin plots show the distribution of DNMT1-Halo in 73–87 cells across 4 replicates. Student t-test, two-tailed, **** P < 0.0001, ** P < 0.01, ns = not significant. (G-I) GSK treatment relocalizes DNMT1 from the nucleoplasm to chromatin. (G) E8 (+/+) cells treated with DMSO for 4 h or with GSK for 4, 24, or 48 h were fractionated into nucleoplasm and chromatin fractions and DNMT1-Halo protein analyzed by JF646 fluorescent gel or immunoblot against FLAG. Note that the levels of DNMT1 in the nucleoplasm decrease following GSK treatment, consistent with total levels shown in (B). PARP1 and histones (from Ponceau gel) are used as protein markers for each compartment. (H) Relative DNMT1 intensity, normalized to total protein loading, from JF646 fluorescent gel. Error bars represent standard error of the mean between 2 and 3 replicates, see Supplementary Fig. S7A and B. (I) DNMT1 intensity in the chromatin fraction divided by that in the nucleoplasmic fraction. Error bars represent standard error of the mean.