Fig. 4: GSK3β inhibitor CHIR potentiates hirsutine-induced 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; the expressions of GSK3β and p-GSK3β were determined by immunoblotting. b A549 cells were pretreated with 5 µM CHIR99021 (CHIR, a GSK3 inhibitor) for 2 h, followed by treatment with 60 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to immunoprecipitation to determine the interaction of p-GSK3β, CypD, and ANT1. c After treatment as indicated in b, the cells were stained with p-GSK3β (Alexa Fluor 647, red), ANT1 (Alexa Fluor 488, green), and DAPI; images were captured by confocal microscope. Scale bar represents 20 μm. d Cells were treated as indicated in b, the calcein fluorescence in the mitochondria was analyzed by microplate reader. e Cells were treated as indicated in b, ATP concentrations were measured by using ATP Determination Kit. f 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