Incubation with 311 decreases cyclin D1 protein half-life and this is mediated by proteasomal activity. (A) 311 decreases cyclin D1 half-life. MCF-7 cells were preincubated with either medium or medium containing 311 (25 μM) for 12 hours at 37°C and then postincubated for 20 to 480 minutes at 37°C with either media alone (control), 311 (25 μM), the protein synthesis inhibitor CHX (10 μg/mL), or CHX (10 μg/mL) and 311 (25 μM). Cells were harvested, and Western blot analysis was performed. (B) The proteasomal inhibitors LC and MG prevented the chelator-mediated decrease in cyclin D1 protein levels, while the DNA-damaging agent Act D (AD) had no effect. MCF-7 cells were incubated for 24 hours at 37°C with either LC, MG, 311, 311 and LC, 311 and MG, AD, AD and LC, AD and LC, or AD and MG. Cells were harvested, and Western blot analysis was performed. Results in panels A and B are representative of 5 and 3 experiments, respectively.