Ostcoarthritis(OA),one of the most common joint discases with unknown etiology,is charac-terized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes.The purpose of this study is to elucidate the molecular mechanisms of H_(2)O_(2)-mediated rabbit chon-drocytes apoptosis.CCK-8 assay showed that H_(2)O_(2) treatment induced a remarkable reduction of cell viability,which was further verified by the remarkable phosphatidylserine extemalization after H_(2)O_(2) treatment for 1 h,the typical characteristics of apoptosis.H_(2)O_(2) treatment induced a signifcant dysfunction of mitochondrial membrane potential(△ψm),but did not induce casapse-9 activation,indicating that H_(2)O_(2) treat ment induced caspase independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently pre-vented H_(2)O_(2)-induced apoptosis.H_(2)O_(2) treatment induced a signifcant increase of caspase8 and-3 activation,and inhibition of caspase-8 or-3 significantly prevented H_(2)O_(2)-induced apoptosis,suggesting that the extrinsic pathway played an important role.Collectively,our findings demonstrate that H_(2)O_(2) induces apoptosis via both the casapse8-mediated extrinsic and the caspaseindependent intrinsic apoptosis pathways in ra bbit chondrocytes.
Fluorescence resonance energy transfer(FRET)technology had been widely used to study proteinprotein interactions in living cells.In this study,we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest(ROI)function of confocal microscope and partial acceptor photobleaching.We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3,and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine(STS)-induced apoptosis.Our results for thefirst demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.
