Fig. 6: ROCK1/PTEN/PI3K pathway regulates hirsutine-mediated GSK3β dephosphorylation, mPTP opening, and apoptosis.

a A549 cells were treated with various concentrations of hirsutine for 24 h or 80 μM hirsutine for different time intervals; whole-cell lysates were prepared and subjected to western blot analysis. CF: cleavage fragment. b, c A549 cells were pretreated with 20 µM Y-27632 (a ROCK1 activation inhibitor) for 2 h, followed by treatment with 80 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to western blot analysis. d Cells were treated as indicated in b, the interaction of p-GSK3β, CypD, and ANT1 was determined by immunoprecipitation. e Cells were treated as indicated in b, the calcein fluorescence in the mitochondria was analyzed by microplate reader. f Cells were treated as indicated in b, ATP concentrations were measured by using ATP Determination Kit. g Cells were treated as indicated in b, C-PARP and C-Caspase 3 in whole-cell lysates were determined by immunoblotting. h Cells were treated as indicated in b, 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