Fig 2: GSK3ß 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 GSK3ß and p-GSK3ß 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-GSK3ß, CypD, and ANT1. c After treatment as indicated in b, the cells were stained with p-GSK3ß (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

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

Fig 4: Reduced mitochondrial calcium buffering and improved bioenergetics in the hippocampi of aged tau-/- mice are reverted by overexpression of CypD. (A) Western blot of hippocampal lysates and (B) densitometric analysis of proteins that form the mPTP, including CypD, ANT, and ATP synthase in aged WT and tau-/- mice. (C) Relative mRNA expression of CypD in aged WT and tau-/- mice. (D) Representation of mitochondrial membrane swelling after calcium overload to determine mPTP opening in aged WT and tau-/- mice. (E) Response of isolated mitochondria after calcium overload. The decreased absorbance indicated mitochondrial swelling. (F) Representation of lentiviral vector transduction in tau-/- mice for the overexpression of CypD [Lenti ORF particles (GFP-tagged)-mouse peptidylprolyl isomerase D (Ppid; cyclophilin D) (#RC223397L2V. Origene)]. (G) Western blot of hippocampal lysates and (H) densitometric analysis of CypD protein in aged WT, tau-/- control, and tau-/- overexpressing CypD mice. (I) Graphical representation of the response of isolated mitochondria after calcium overload in aged WT, tau-/- control, and tau-/- CypD mice. (J) ATP concentrations measured in whole hippocampal extracts from aged WT, tau-/- and tau-/- CypD mice. ATP concentration is expressed as pmol of ATP/µg of total protein extract. *p < 0.05, **p < 0.01; mean ± S.E.M.

Fig 5: PFOS induces mitochondrial dysfunction and apoptosis in macrophages.a, b Bone marrow-derived macrophages (BMDMs) were treated with PFOS (150 µM, 6 h), then the cells were stained with Mitotracker deep red (a) or tetramethylrhodamine methyl ester (TMRM) (b) to detect the mitochondrial respiration and membrane potential by flow cytometry, respectively. c Immunoblot analysis of Cytochrome c release and apoptosis in BMDMs as indicated treatment. d, e THP-1-derived macrophages were treated with DMSO or cyclosporine A (50 µM, red plots) for 1 h followed by PFOS (150 µM, 6 h), poly (dA:dT) (2 µg/ml, 6 h) treatment or pre-treated with LPS (200 ng/ml, 3 h) followed ATP (5 mM, 6 h) treatment. IL-1ß secretion in the supernatants of the indicated THP-1-derived macrophages were determined by ELISA (d). The caspase-1 activation (p10) and IL-1ß maturation (p17) in the cell supernatants or pro-caspase-1 and pro-IL-1ß in the cell lysates were detected by immunoblot (e). f Immunoblot analysis of the supernatants and cell extracts of BMDMs pre-treated with DMSO or cyclosporine A (50 µM) for 1 h. BMDMs were then treated with PFOS (150 µM, 6 h), poly (dA:dT) (2 µg/ml, 6 h) or pre-treated with LPS (200 ng/ml, 3 h) followed ATP (5 mM, 6 h) treatment. g, h Wild type (WT, black plots), CYPD knockout (KO#1: red plots; KO#2: blue plots) THP-1-derived macrophages were treated with PFOS (150 µM) for 6 h. Relative enrichment of mtDNA in AIM2-pulldown material were determined (g). IL-1ß production in the supernatants were measured by ELISA (g). The maturation of IL-1ß in the supernatants or pro- IL-1ß and CypD in lysates were detected by immunoblot (h). i, j Immunoblot analysis of Cytochrome c from cytosolic extracts and mitochondria from indicated cells pre-treated with DMSO or cyclosporine A (50 µM) for 1 h, followed with PFOS treatment (150 µM, 6 h) (i). Relative enrichment of mtDNA in AIM2-pulldown material from the THP-1-derived macrophages (WT, black plots; CYPD KO#2: blue plots) were checked (j). In a, b, d, g and j, all error bars, mean values ± SEM, P-values were determined by unpaired two-tailed Student’s t test of n = 3 independent biological experiments. For c, e, f, h and i, similar results are obtained for three independent biological experiments. Source data are provided as a Source data file.