SCF^Skp2 inhibition induces caspase-independent apoptosis and cell-cycle arrest. (A) A panel of myeloma cell lines was cultured in the presence of varying concentrations of CpdA for 3 days. Cell growth was assessed using the WST-1 assay, and the concentration that induced IC₅₀ was calculated. The data shown represent the means plus or minus SD of triplicate cultures. (B) RPMI 8226 cells were treated with vehicle or 5 or 10 μM CpdA for 24 hours. They were then stained with propidium iodide and analyzed by flow cytometry to determine the cell-cycle distribution. (C) RPMI 8226 cells were treated with either 10 nM bortezomib or 5, 10, or 15 μM CpdA, and then analyzed for externalization of phosphatidyl-serine by staining with annexin V. After analysis by flow cytometry, the proportion of specific apoptosis is indicated as the mean plus or minus SD from triplicate experiments. Specific apoptosis was calculated as ((% annexin V⁺ cells in drug-treated samples − % annexin V⁺ cells in vehicle-treated controls) / (1 − % annexin V⁺ cells in vehicle-treated controls)) × 100. (D) Bortezomib- or CpdA-treated RPMI 8226 cells were probed for the presence of full-length and cleaved caspase-8, caspase-9, caspase-3, and PARP by Western blotting. (E) RPMI 8226 cells were preincubated with vehicle or 50 μM PAN-caspase inhibitor for 2 hours, and then treated with vehicle, CpdA, or bortezomib. Cell viability was determined using the WST-1 assay, and is shown as a mean value plus or minus SD from triplicate experiments. (F) Cells treated as described in panel E were analyzed by flow cytometry after staining with annexin V–PE and FITC-VAD-FMK to identify viable cells (PE⁻/FITC⁻), as well as those undergoing apoptosis (PE⁺). The proportion of cells present in the respective quadrants is indicated inside each of the panels. Data are presented from 1 of 3 independent experiments.