Fig 4.

ALA blocks Pi-induced oxidant generation in VSMCs and decelerates Pi-induced VSMC calcification by inhibiting apoptosis and stimulating survival. (A, B) ALA suppresses Pi-induced oxidant production and thiol oxidation in VSMCs. After VSMCs were treated with Pi in the presence or absence of ALA (300 μM) or antimycin A (AA, 20 μM), an oxidant generator by inhibiting mitochondrial complex III, for 4 days, oxidant formation was assessed by confocal microscopy using the fluorescent probe DCFH-DA (A) and intracellular thiol content was measured (B). The fluorescence index was represented as the collected values of fluorescence intensity for the section of photography using Image-Pro Plus version 6.0. Scale bar indicates 100 μm. Calcium content was determined (C). Data in (B) and (C) are expressed as the mean ± S.D. (n= 3). (D, E) After VSMCs were treated with combination between Pi, ALA and AA for 4 days, release of Cyt C from mitochondria to cytosol (D) and cell survival Gas/Axl signal in whole cell extracts (E) were analysed using Western blotting with specific antibodies to Cyt C, Cox-2, Gas6, Axl and β-actin. *P < 0.01; ^†P < 0.05.