Figure 5.

ISM interacts with AACs in mitochondria and blocks ATP transport from mitochondria to cytosol. (a) rISM co-IP with AACs in mitochondrial fraction of HUVECs. (b) rISM treatment reduced ATP level in cytosolic fraction of HUVECs independent of caspases activity. Z-VAD-fmk was used as pan-caspase inhibitor. **P<0.01, n=3. Error bars denote S.E.M. (c) rISM treatment increased ATP level in mitochondrial fraction of HUVECs independent of caspases activity. Z-VAD-fmk was used as pan-caspase inhibitor. **P<0.01, n=3. Error bars denote S.E.M. (d) rISM treatment diminished ATP concentration in whole-cell lysates of HUVECs independent of caspases activity. Z-VAD-fmk was used as pan-caspase inhibitor. *P<0.05, n=3. Error bars denote S.E.M. (e) rISM treatment did not affect the mitochondrial potential of HUVECs. The UV-treated HUVECs were used as the positive control for the loss of mitochondrial potential via the intrinsic apoptotic pathway. The untreated HUVECs were used as the negative control. MitoTracker Green can stain mitochondria regardless of mitochondrial potential. TMRM (red) was used as the marker of mitochondrial potential. Nuclei were labeled by Hoechst (blue). (f) rISM treatment did not induce the release of cytochrome c and AIF from mitochondria to cytoplasm. The upper panel is the cytosolic fraction without mitochondria of HUVECs. The lower panel is the mitochondrial fraction. The UV-treated HUVECs were used as the positive control for the release of cytochrome c and AIF from mitochondria to cytoplasm via the intrinsic apoptotic pathway. β-actin was used as the marker for cytosolic fraction. VDAC was used the marker for mitochondria. (g) Schematic summary of the putative proapoptotic pathway engaged by ISM-GRP78 interaction