Figure 6.

Mechanism of Cytotoxicity. (A) Lack of a Role for Caspases. Cells (75,000/well) were treated with UNC7938 or staurosporine for 2 h, both in the presence of 10 or 30 μM QVD a pan–caspase inhibitor. After removal of the test compounds incubation was continued overnight in the presence of QVD. Thereafter the Alamar Blue cytotoxicity assay was performed. Means ± SE. N = 3. Left panel: UNC7938. Right panel: staurosporine. (B) Lack of Effect of a Lysosomal Protease Inhibitor. Cells (75,000/well) were treated with UNC7938 for 2 h in the presence of 10 or 30 μg/ml E64d, a cathepsin inhibitor. After removal of the test compound incubation was continued overnight in the presence of E64d. Thereafter the Alamar Blue cytotoxicity assay was performed. Means ± SE. N = 3. (C) OEC Effects on Plasma Membrane Permeability. Cells were incubated with various concentrations of UNC7938 for 2h in medium, rinsed in PBS, and then incubated with 4 μM ethidium dimer and 2 μM Calcein AM in PBS for 30 min. Cells were rinsed twice in PBS and then lysed in 0.3 ml of 0.2% TX100 in PBS. Ethidium and calcein fluorescence was measured using the appropriate settings on a plate reader. The ethidium/calcein ratio, normalized to untreated controls, is shown. Means ± SE. N = 6. The inset depicts cells permeabilized with the detergent TX100 as a positive control. (D) OEC Effects on Plasma Membrane Permeability Continued. Cells were co-incubated in medium with 4 μM ethidium dimer and various concentrations of UNC7938 for 2 h. Cells were rinsed twice in PBS and then lysed in 0.3 ml of 0.2% TX100 in PBS. Ethidium fluorescence was measured using a plate reader. The ethidium/cell protein ratio, normalized to untreated controls, is shown. Cytotoxicity measured using the Alamar Blue assay is also shown. Means ± SE. N = 3.