Figure 3.

Dynamics of DNMT1 through the S phase of the cell cycle. (A) Double thymidine block protocol used to synchronize cells to S phase. (B) Flow cytometry after propidium iodide staining to show cell cycle progression (based on the DNA content of the cells). Following 1 h post-thymidine release, there is an increase in S phase cells which culminates at 3–5 h post-release. (C-I) Cells were synchronized using a double thymidine block and labelled as described in text. Spot-on analysis showing the dynamics of DNMT1-Halo molecules. (C) Pie charts showing the distribution of fast or slow diffusing and chromatin-bound DNMT1 molecules at 1 and 5 h post-thymidine release. (D-I) Violin plots showing the distribution of DNMT1-Halo in 62-152 cells across 2–5 replicates. (D and E) and (G and H) For fastest and slower diffusing molecules, mid-S phase cells (5 h post-thymidine release) have less mobile DNMT1-Halo compared to cells at G1/S (1 h). (F) and (I) The fraction of DNMT1-Halo tagged molecules that are chromatin bound increases in mid-S phase (5 h) compared to G1/S (1 h) and G2 phase (9–10 h) of the cell cycle. Student t-test, two-tailed, **** P < 0.0001, *** P < . 0.001, ns = not significant.