Fig. 4: Respiratory dysfunction mediated by NCLX loss in BA is acutely induced following adrenergic stimulation.

a, b Western blot analysis of NCLX-null and WT BA for UCP1 expression levels S.F. Gel was used as a normalization reference for total protein loading (n = 11 per group). c, d Western blot analysis of NCLX-null and WT BA for TOM20 expression levels (n = 7 per genotype). e Mitotracker Green (MTG) fluorescence intensity in NCLX-null and WT primary BA, measured by Operetta high-content imaging system (n = 16 per condition from total N = 3 imaging experiments). f–j Membrane potential and morphometric analyses of mitochondrial morphology in NCLX-null and WT BA (N = 4 independent experiments and n = at least 41 cells per condition). f Representative images of NCLX-null and WT BA stained with TMRE and MTG. Scale bar, 10 μm. g Quantification of mitochondrial membrane potential of NCLX-null and WT BA. TMRE signal is normalized to MTG signal to generate a ratio that avoids focal plane artefacts. h Quantification of mitochondrial aspect ratio (AR), i circularity and j roundness in NCLX-null and WT BA using the MTG channel. k, l Western blot analysis of primary NCLX-null and WT BA for OXPHOS complex subunits II–V (CII–CV). Cells were treated with or without NE for 6 h (n = 3–7 per group). m Western blot of super complex (SC) I+III in BAT homogenates from cold-exposed and non-exposed NCLX-null and WT mice. n Quantification of the fraction of complex III that is assembled into SC(I+III) relative to total complex III from cold-exposed and non-exposed NCLX-null and WT mice (N = 3–4 mice per condition). o Assessment of mitochondrial complexes activity in cold-exposed and non-exposed NCLX-null and WT BAT: CI (n = 11–18), CII (n = 9–20), and CIV (n = 5–8). p Quantification of basal respiration in intact non-stimulated BA (N = 6–8 independent experiments with n = 32 for WT and n = 21 for NCLX KO). q Quantification of maximal spare respiratory capacity in NE-stimulated and non-stimulated NCLX-null and WT BA. Maximal respiration was induced by FCCP (N = 3–8 independent experiments with n = 18–32 per condition). Student’s t-test (b, d, e, g, h, i, j, p); one-way ANOVA with Tukey’s post hoc test (l, n, o, q). Data are expressed as means ± SEM. ^nsp > 0.05, **p < 0.001. Replicates are indicated by individual dots shown for each group in all graphs. Source data are available as a Source Data file.