Figure 2.

Effect of spermidine on extrinsic and intrinsic pathways in H₂O₂-induced apoptosis. Cells were pre-treated with or without 10 μM spermidine for 1 h and then additionally incubated with 300 μM H₂O₂ for 24 h. (A) The expression of extrinsic and intrinsic apoptosis-regulatory proteins was evaluated by Western blot analysis with whole cell lysates. Equal protein loading was confirmed by β-actin as an internal control. (B,C) Flow cytometry: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanune iodide (JC-1). (B) Representative histograms. (C) The percentages of monomers were determined by counting the percentage of JC-1 green-positive cells. Data are expressed as the mean ± SD (n = 3). *** p < 0.001 when compared to control. ## p < 0.01 when compared to H₂O₂-treated cells. (D) Cells probed with 100 nM MitoTracker Red and observed under a fluorescence microscope. Scale bar: 200 μm. (E) Cytosolic and mitochondrial proteins were isolated and the expression of cytochrome c was detected by Western blot analysis. Cytochrome oxidase subunit VI (COX IV) and β-actin served as protein loading controls for the mitochondria and cytosol, respectively. The activities of caspase-3 (F), caspase-8 (G), and caspase-9 (H) were measured using caspase colorimetric assay kits. Data are expressed as the mean ± SD (n = 3). * p < 0.05 and *** p < 0.001 when compared to control. ## p < 0.01 and ### p < 0.001 when compared to H₂O₂-treated cells.