Normalization of histone acetylation status and cell cycle progression upon SFN removal. (A) HCT116 cells were treated with 15 μM SFN as described in Figure 3 legend, using 6-h, 24-h, and continuous exposure protocols. At 48 h after SFN was first added to the cells, whole cell lysates were prepared and subjected to immunoblotting for total histone H4 (H4), H4K12ac, p21WAF1, and β-actin. (B) The cell cycle distribution was determined after 72 h using flow cytometry (see Methods), for HCT116 cells treated with 15 μM SFN continually, or for 24 h and replaced with fresh media containing no SFN. (C) The experiment in (B) was repeated three times and the percent of cells in G0/G1, S, and G2/M was quantified. Data (mean ± SE, n = 3); *P < 0.05, **P < 0.01 versus the corresponding DMSO control. (D) HCT116 cells were treated with 15 μM SFN continually or for 24 h and replaced with fresh media (no SFN), and the corresponding whole cell lysates were immunoblotted at 48 or 72 h for full-length poly(ADP-ribose)polymerase (PARP), or its cleavage product (arrow). Results are representative of the findings from two or more separate experiments.