FIG. 8.
Cell cycle analysis after PR-Set7 depletion. (A) Experimental design. PR-Set7flox/−; CreERT cells were synchronized with NCZ for 12 h followed by treatment with thymidine in the presence or absence of 4-OHT for 24 h and released into fresh ES medium. Samples were collected for FACS analysis and protein extraction at the time points shown at the top (black triangles). (B) Changes of the level of PR-Set7 protein and H4K20me1. Shown are Western blot analyses using the antibodies shown to the left and nuclear protein isolated from the cells at the time points indicated at the top. PR-Set7 protein was undetectable at 0 h after 24 h of treatment with 4-OHT (bottom). Both β-tubulin and histone H4 are shown as loading controls. (C) FACS analyses of the cells at each time point as indicated at the top. Note that cells treated with 4-OHT (bottom) show slower cell cycle progression between 8 h and 12 h than the cells untreated with 4-OHT (top) and accumulate at early S phase at 12 h (bottom right). AS, asynchronous.