Apoptosis induction by GCS-100 is associated with a time- and dose-dependent loss of mitochondrial transmembrane potential and caspase cleavage. (A-B) RPMI 8226 (▴) and U266 (■) were incubated with GCS-100 (0-800 μg/mL) for 48 hours. Mitochondrial transmembrane potential was assessed by examining uptake of DiOC₆(3) by flow cytometry, where low DiOC₆(3) indicates loss of transmembrane potential. The proportion of cells with low DiOC₆(3) (A) and the fraction of DAPI-negative cells with low DiOC₆(3) (B) are displayed. Results are the mean ± SEM of triplicate experiments. *Significant difference compared with nonexposed cells (P ≤ .05). (C) RPMI 8226 (▴) and U266 (■) cells were cultured with 500 μg/mL (RPMI 8226) or 800 μg/mL (U266) GCS-100 for up to 72 hours followed by mitochondrial transmembrane potential assessment using DiOC₆(3). Results are the mean ± SEM of 3 independent experiments. *Significant difference compared with nonexposed cells (P ≤ .05). (D) Western blot analysis. RPMI 8226 cells were cultured with 500 μg/mL GCS-100 for up to 48 hours. Whole-cell lysates were prepared, and 50 μg of protein was resolved by 12% SDS-PAGE. Protein was transferred to PVDF membrane and probed with anticaspase-9, anticaspase-8, and anticaspase-3 antibodies that detect unprocessed and cleaved protein (←). Molecular weight markers are shown on the left. Representative results of a minimum of 2 independent experiments are shown.