Figure 8.

Effect of cholesterol addition on Rh2-induced cell death. (A) Serum-starved A431 cells were treated either with 30 µM Rh2, 30 µM cholesterol, or 300 µM cholesterol. Some Rh2-treated cells for 4 h were incubated with either 30 µM cholesterol or 300 µM cholesterol for additional 4 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. (B) Serum-starved A431 cells were treated either with 30 µM Rh2 for 4 h or 8 h. Some Rh2-treated cells were with 300 µM cholesterol for 1 h or 4 h and processed for immunoblotting analysis using anti-phospho-Akt and anti-β-actin antibodies. (C) A431 cells were treated as described in Figure 8A and subjected to flow cytometer analysis of annexin V-FITC and propidium iodide (PI)-stained cells. Annexin V-FITC only (early apoptosis) and positive for both annexin V-FITC and PI (late apoptosis) were quantitated, and both subpopulations were considered as overall apoptotic cells. These experiments were performed twice with comparable results.