Fig. 3: Hirsutine induces apoptosis dependent on mitochondrial permeability transition pore (mPTP) opening.

a, b A549 cells were pretreated with 5 µM cyclosporin A (CSA, a CypD inhibitor) for 2 h, followed by treatment with 80 μM hirsutine for 24 h. Cells were stained with calcein-AM and CoCl₂ (cytosolic calcein quencher), the calcein fluorescence in the mitochondria was analyzed by confocal laser scanning microscope or microplate reader. Scale bars: 20 μm. Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01. c Cells were treated as indicated in a, equal amount of lysates were subjected to immunoprecipitation using anti-ANT1 antibody, the associated CypD was determined using immunoblotting. d Cells were treated as indicated in a, the ATP concentrations were measured using a firefly luciferase-based ATP Determination Kit. e Cells were treated as indicated in a, the percentage of apoptotic cells was determined by flow cytometry using Annexin V-FITC/PI staining. Data are expressed as the mean ± SD (n = 3), **P < 0.01