Effects of NaHS on mitochondrial function.

<p>(<b>A</b>) ATP synthesis. After pretreatment with vehicle, 300 μM NaHS or 10 mM PAG for 6 hours and followed by exposure to 600 μM H₂O₂ for another 4 hours, HUVECs were harvested to collect mitochondria. The rate of ATP synthesis was expressed by µmol ATP/min/g of mitochondrial protein. (<b>B</b>) Release of cytochrome c from mitochondria. After treatments as previous description, HUVECs were harvested to collect mitochondria and cytosol. The protein expression was tested by western blot. The bar chart showed the ratio of cytochrome c in cytosol to that in mitochondria, indicating the intensity of release of cytochrome c. (<b>C</b>) MDA changes in HUVECs mediated by H₂O₂. The data are expressed at nmol/mg. (<b>D</b>) Fluorescent intensity of DPPP in HUVECs mediated by H₂O₂. (<b>E</b>) ROS production was stained by 10 μM H₂DCFDA for 20 min, whose oxidation product (DCF) fluorescence indicated ROS formation. (<b>F</b>) ROS production was stained by 5 μM DHE for 30 min, which fluorescence indicated ROS formation. The absorbance values in (D)–(F) of HUVECs were normalized against the values for normal controls and expressed as a percentage of control. (<b>G</b>)–(<b>L</b>) JC-1 staining. Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of Δ<i>Ψ</i>_m. (G)–(J) HUVECs were stained with JC-1. (K) CCCP was the positive control. Cells were observed under ×200 microscopy. Scale bar is shown at 100 μm. (L) Ratio of red to green fluorescence, indicating ratio of JC-1 polymer/monomer. The data shown are mean ± SEM (n = 6). * <i>p</i><0.05, ** <i>p</i><0.01vs control. # <i>p</i><0.05, ## <i>p</i><0.01 vs vehicle + H₂O₂ group.