6.
Adenoviral-mediated overexpression of Bcl-xL reduces hypoxiaand doxorubicin-induced H9c2 cell apoptosis. Cells were transduced with AdBcl-xL or AdNull at an MOI of 1000 for 1 hr and cultured for 24 hrs before treatment with hypoxia and doxorubicin (0.5 μM) for 48 hrs. (A) Overexpression of Bcl-xL confirmed by Western blotting in samples 24 hrs after transduction. (B) Hypoxia- and doxorubicin-induced accumulation of cytochrome c in the cytosol is prevented by overexpression of Bcl-xL. Subcellular fractionation was performed by differential centrifugation of cell lysates exposed to hypoxia or doxorubicin with the cytosolic-rich fractions analysed for presence of cytochrome c by Western blotting. Bcl-xL prevents pro-caspase-3 processing induced by hypoxia and doxorubicin. Whole cell lysates of nontransduced, AdNull- and AdBcl-xLtransduced cells, untreated and treated, were analysed for pro-caspase-3 cleavage by Western blotting. (C) Non-transduced, AdNull and AdBcl-xL infected cells were exposed to hypoxia for 48 hrs or doxorubicin for 24 hrs and stained with Hoechst to analyse nuclear fragmentation. Overexpression of Bcl-xL protects cells from hypoxia- and doxorubicin-induced apoptosis.