Fig. 5: Adrenergic stimulation in NCLX-null BA leads to mitochondrial swelling and cytochrome c release.

a–c Super-resolution confocal images of fixed WT or NCLX-null BA, with or without adrenergic stimulation for 6 h. a NCLX KO and WT BA treated with or without NE and immunostained for cytochrome c (red). Samples were co-stained for TOM20 to label mitochondrial localization. Scale bar is 10 μm, for the zoom images scale bar is 2 μm. b Quantification of cytochrome c release. Cytochrome c in the cytosol was calculated by measuring its total integrated fluorescence intensity (FI) outside the mitochondria per cell, normalized to total cellular area (n = 27–46 cells for each of the four conditions). c Quantification of mitochondrial swelling. Zero indicates bars where quantification yielded zero events of swelling (n = 28–37 cells for each of the four conditions). d Time-lapse of swelling formation following NE stimulation in NCLX-null and WT BA. Cells were labeled with mCherry-Fis1 probe marking their outer mitochondrial membrane. Swelling spatially starts at one pole of the cell and gradually propagated to the center of the cell. Scale bar, 10 and 2 μm for the zoom images. e–h Electron microscopy of NE-stimulated NCLX-null and WT BA. e Representative images of NE-stimulated NCLX-null and WT mitochondria of primary BA. Note that while the WT mitochondria are intact, adrenergic stimulation leads to cristae disruption, mitochondrial swelling, and rupture in the NCLX KO mitochondria. Scale bar, 1 and 0.1 μm for the zoom images. f Quantification of cristae density. g Circumference and h mitochondrial swelling assessed by cross-sectional area of individual mitochondria (n = 185 mitochondria at least were analyzed per condition). Student’s t-test (f, g, h); one-way ANOVA with Tukey’s post hoc test (b, c). Data are expressed as means ± SEM. ^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.0001. Replicates are indicated by individual dots shown for each group in all graphs. Source data are available as a Source Data file.