P-V blocks the mitochondrial membrane potential and induces mitochondrial cell death: The role of mitochondrial STAT3.

<p>(<b>A</b>) Knocking-down STAT3 increases mitochondrial O₂⁻ levels in BxPC-3 cells. Control-siRNA and STAT3-siRNA cells were preloaded with MitoSOX Red, and mitochondrial O₂⁻ was determined by flow cytometry (<i>left</i>) or confocal microscopy (<i>right</i>; x40). (<b>B</b>) STAT3 overexpression reduces the increase in mitochondrial O₂⁻ induced by P-V. BxPC-3 cells were transfected with a STAT3-expressing plasmid or a control plasmid (empty vector) for 48 h, and then treated with P-V for 4 h followed by addition of the probe MitoSOX Red. Cells were then examined by confocal microscopy (x40). (<b>C</b>) P-V collapses the mitochondrial membrane potential (ΔΨm) in a time- (<i>top</i>) and concentration-dependent (<i>bottom</i>) manner. Fluorescence histograms were quantified and results are shown as mean±SEM; *<i>p</i><<i>0.05 vs</i>. control. (<b>D</b>) Immunoblots for cytochrome c, pro-caspases and caspases 9 and 3 in cytosolic protein extracts from BxPC-3 cells treated with P-V. (<b>E</b>) BxPC-3 mitochondria-less (ρ⁰) cells are markedly more resistant to P-V-induced apoptosis. <i>Top</i>: Immunoblots for COX IV and STAT3 in parental (−) and ρ⁰ BxPC-3 cells. Loading control: β-actin.