FIGURE 2.

NSP4 depolarizes mitochondria and induces release of mitochondrial cyt c. A, effect of NSP4 on mitochondrial membrane potential. 293T cells were transfected with pcDNSP4 or vector control, and 18 h post-transfection, cells were incubated with 100 nm TMRE for 30 min at 37 °C followed by analysis for TMRE accumulation within mitochondria using flow cytometry. Compared with vector control (I), pcDNSP-expressing cells showed significant dissipation of mitochondrial potential (II). In cells pretransfected with Bax-siRNA followed by transfection with pcDNSP4 (IV) or pcDNSP4-expressing cells treated with 50 μm BAPTA-AM, (6 h post-transfection) (VI), depolarization of mitochondria was ≈20–40% less compared with only pcDNSP4-expressing cells (II). In the presence of both Bax-siRNA and BAPTA-AM (VIII), significant reduction (50%) in NSP4-mediated dissipation of mitochondrial potential (II) was observed. B, IVT NSP4 depolarized purified mouse mitochondria. Mouse liver mitochondria (50 μg of total protein) were incubated for 10 min at RT with IVT NSP4 (0–0.2 μm) followed by incubation with TMRE (50 nm) for 10 min at RT, followed by measurement of mitochondrial membrane potential dissipation fluorometrically. Data are represented as percent fluorescence relative to mock-treated mitochondria. C, cyt c released by NSP4 is partly independent of Ca²⁺ ion and Bax activation. 293T cells were transfected with either Bax-siRNA or nonspecific control siRNA. After 24 h, both sets of cells were transfected with pcDNSP4 or pcDNA control. After 6 h, cells were treated with BAPTA-AM (50 μm). After 24 h of transfection, cells were harvested, and subcellular fractions were separated followed by immunoblotting of cytosolic extract with cyt c antibody. Pellet was immunoblotted with Cox4 and β-actin as control. D, IVT NSP4 also releases cyt c from mouse liver mitochondria. Mouse liver mitochondria (50 μg) were incubated with IVT NSP4 (0–0.2 μm) for 1 h at 30 °C. Reaction mixture was centrifuged (7000 × g for 10 min) to pellet down mitochondria, and supernatants (supe) was immunoblotted to assess cyt c release in cytosol. IVT reaction mixture control and IVT pcDNA control were used as negative control. Mitochondrial pellet was immunoblotted with anti-Cox4 antibody as internal control. E, NSP4-induced cyt c release is independent of caspase activation. 293T cells transfected with pcDNSP4 cells were either treated with broad spectrum caspase inhibitor Z-VAD-fmk (10μM) or DMSO control followed by subcellular fractionation after 24 h. Cyt c release into cytosol was detected by immunoblot analysis. F, NSP4-induced cell death is dependent on caspase activation. 293T cells transfected with pcDNSP4 cells were either treated with broad spectrum caspase inhibitor Z-VAD-fmk (10 μm) or DMSO control followed by cell death analysis by TUNEL assay.