Figure 4

Induction of apoptosis with T0901317 treatment. Flow cytometric analysis of apoptosis was utilized for determination of caspase-3 and -7 activities. CaOV3 cells were treated with either vehicle (DMSO) or T0901317 at the indicated doses (10 μM to 40 μM) for 24 hours and then stained with Vybrant FAM dye, and 7-AAD strictly following manufacturer's instruction. Data are mean ± SD of three different experiments (A). Caspase 3/7 activity was also measured in CaOV3 cells after 12 hours of treatment with vehicle (DMSO) or 5 μM, 10 μM, 20 μM, 40 μM or 50 μM. A luminescent assay was used, as detailed in Material and Methods. T0901317 significantly increases Caspase 3/7 activation. Results are the mean ± SD of three independent experiments and are expressed as percentage of negative control (DMSO). (* p < 0.006 vs. negative control, (B). The activation of caspase 3 was confirmed by Western Blot analysis. LXR agonist treatment enhances caspase 3 activation, resulting in increased caspase 3 precursor cleavage rate and decreased caspase 3 precursor protein levels. Decreased caspase-3 precursor protein levels occur in a concentration dependent manner (C). β-actin expression was determined by Western blot analysis and used as an endogenous control.