Ouabain-induced caspase-dependent apoptosis in LN229 cells. Ouabain-induced cell death and apoptotic signaling were evaluated using flow cytometry and Western blot analysis. A. Flow cytometry results showed that ouabain treatment (1 μM, 5 hrs) increased early (Q3 quadrant) and late apoptotic (Q2 quadrants) cell populations. N = 3 independent assays. B, C, and D. Western blot analysis revealed that ouabain treatment of 3 hrs induced significant activation of caspase-3 and caspase-9 in LN229 cells. The caspase activations subsided thereafter. E. Western blotting indicated that although the anti-apoptotic gene Bcl-2 showed a trend of increasing at an early stage during ouabain exposure, the Bcl-2 level significantly decreased 24 hs after ouabain treatment. N = 3 in each group. F. Ouabain-induced cell death in LN229 cells was partially blocked by co-applied caspase inhibitor Z-VAD (100 uM). Cell death was measured using the MTT assay. Three independent experiments and each group in an experiment contained 3–4 duplicates. * P < 0.05 vs. control (CTL).