Fig. 8.

Deacetylation of DNMT1 affects its cell cycle regulatory function. (A) 293T cells were transfected with GFP-SIRT1 and either wild-type or mutant HA-DNMT1 expression plasmids. Cell lysates were immunoprecipitated with antibody to HA, and precipitated material was immunoblotted with antibodies to GFP or HA. GFP-SIRT1 and HA-DNMT1 expressions were monitored by direct Western blotting with anti-GFP and anti-HA antibodies, respectively. (B) Wild-type HCT116 (WT) cells or DNMT1-knockout (KO) HCT116 cells were cotransfected with GFP and either wild-type DNMT1 (WT), mutant HA-DNMT1, or vector plasmid. The ratio of GFP to HA-DNMT1 was 1:10. Cells received 10 Gy of gamma irradiation (IR) 36 h after transfection and were harvested at 48 h posttransfection. Cell cycle position was determined by FACS analysis of propidium iodide-stained cells for GFP-positive cells. The data shown are the average values ± standard deviations from three separate experiments (right).