Figure 7.
SIN-1 attenuates staurosporine-induced caspase-3 activation and apoptotic events, but not cell death. A, Caspase-3 immunoblot of whole-cell lysates taken at the indicated times after treatment with 1 μm staurosporine alone or in combination with 1 mm SIN-1. Active caspase-3 immunoreactivity (∼17 kDa band) induced by staurosporine (lanes 2–5) was abolished after the application of 1 mm SIN-1 (lanes 6–9). B, Caspase-3 activity measurements with Ac-DEVD-AMC using cell lysates taken (24 h) after the indicated insults (n = 4 experiments; asterisk, different from control at p < 0.05). C, Effect of staurosporine alone and in combination with SIN-1 on PARP-1 cleavage (n = 2 experiments; +Ve indicates positive control using camptothecin-treated Jurkat cells). D, Staining of the cultures using FITC-conjugated annexin V, a label of inverted phosphatidylserine, after treatment with staurosporine alone or in combination with SIN- 1 (n = 3 experiments). Scale bar, 50 μm. For quantification, FITC fluorescence was averaged from several images taken using identical settings, normalized to baseline controls, and expressed as a percentage increase over baseline controls. The application of 1 mm SIN-1 significantly reduced FITC staining compared with staurosporine treatment alone (p < 0.05; n = 4 experiments per condition). E, TUNEL (green) and Hoechst (red) nuclear labeling of cultures after treatment with staurosporine alone or the staurosporine/SIN-1 combination (n = 3 experiments per condition). Scale bar, 100 μm. No significant difference (see Results) is observed in cell death after the application of 1 mm SIN-1 compared with staurosporine alone (also compare images with Fig. 5C).
