Fig. 6: Adrenergic stimulation in NCLX-null BA leads to Ca²⁺-mediated cell death in vitro and in vivo.

a–c Ca²⁺ chelation and inhibition of Ca²⁺ uptake prevent mitochondrial swelling in NCLX KO BA. a A scheme depicting points of pharmacological intervention targeting Ca²⁺ entry and accumulation. EGTA (3 mM) was used to buffer extracellular Ca²⁺, BAPTA-AM (25 μM) was used to chelate intracellular Ca²⁺, and Ru360 (10 μM) was used to inhibit MCU and Ca²⁺ entry to the mitochondria. b Quantification of mitochondrial swelling in NE-stimulated NCLX KO and WT BA treated with Ca²⁺ chelators or Ru360 assessed by super-resolution imaging of TOM20 staining for mitochondrial outer membrane (n = 18–33). c Representative images of NE-stimulated NCLX KO and WT BA treated with Ca²⁺ chelators or Ru360. Note that chelating Ca²⁺ or blocking mitochondrial Ca²⁺ entry was sufficient to inhibit mitochondrial swelling. Scale bar, 10 and 1 μm for the zoom images. Swollen mitochondria are indicated by the white arrows. d–f Cell death measured by TUNEL staining. Mice were submitted to intermittent cold-stress protocol for 5 days. BAT was harvested, fixed, and stained with TUNEL. Hematoxylin was used as a counterstaining for all nuclei (n = 3–6 mice per genotype). d TUNEL staining of BAT lobes from cold-stressed NCLX KO and WT mice. TUNEL-positive cells (brown) are indicated with red arrows. Scale bar, 500 and 50 μm for the zoom images. e Quantification of the average number of positive cells per cross-sectional area in each genotype. f Quantification of the TUNEL-positive cells as a percentage of total cells. Student’s t-test (e, f); one-way ANOVA with Tukey’s post hoc test (b). 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.