Fig 3.

ALA inhibits mitochondria-mediated apoptosis of VSMCs induced by Pi and restores Gas6-Axl survival pathway. (A–C) Inhibition of Pi-induced VSMC apoptosis by ALA. VSMCs were treated with Pi in the presence or absence of ALA (300 μM) for 4 days (A, C) or indicated times (B). Apoptotic cells were detected by TUNEL staining (A). Nuclei were counterstained with propidium iodide and then the two images were merged. TUNEL-positive apoptotic cells were counted as a percentage of the total number of cells. Scale bar indicates 100 μm. To assess the release of cytochrome c from mitochondria into cytosol (B), mitochondrial and cytosolic fractions were subjected to Western blotting with antibodies against cytochrome c (Cyt C), cytochrome c oxidase 2 (Cox 2, a mitochondrial marker protein) and β-actin (a cytosolic marker protein). Cytosolic extracts were analyzed for caspase-9 or caspase-3 activity. The activity was normalized to protein content and expressed as a fold difference relative to control (C). Data are expressed as the mean ± S.D. (n= 3). (D) ALA restored Gas6 and Axl expression and Akt activation, which are down-regulated by Pi. Whole cell lysates were obtained from VSMCs treated with Pi for the indicated times and subjected to Western blotting with specific antibodies to Gas6, Axl, β-actin, phospho-Akt and Akt. *P < 0.01; ^†P < 0.05.