Probing the pathways of protein turnover and stability in SFN-treated colon cancer cells. (A) HCT116 cells were treated with MG132, N-acetyl-Leu-Leu-norleucinal (ALLN), leupeptin, or PYR-41 , in the presence and absence of SFN (15 μM). Whole cell lysates obtained at 24 h were immunoblotted for HDAC3. For the concentrations of each inhibitor, see Methods. (B) HDAC activity in the whole cell lysates obtained at 24 h from HCT116 cells treated with DMSO, SFN, PYR-41 (PYR), or SFN+PYR. Data (mean ± SE, n = 3); **P < 0.01 versus the DMSO control. (C) HCT116 cells were treated with inhibitors, as shown, and the whole cell lysates were immunoblotted at 24 h for total cellular ubiquitin. (D) HCT116 cells were treated with cycloheximide (CHX), actinomycin D (Act D), SFN, SFN+CHX, or SFN+Act D. Whole cell lysates were immunoblotted at 6 h for HDAC3, HDAC4, SMRT, N-Cor and p21WAF1. Data are representative of findings from two or more separate experiments.