(A and B) mtROS were detected by MitoSOX in iBAT. Data are presented as arbitrary fluorescence unit (AFU). n = 5. (C) mtROS were detected by MitoSOX (red) and mitoPY1 (green) in freshly isolated iBAT. Data are presented as relative fluorescence unit (RFU) by taking WT as 1.0. n = 6. (D) Western blot analysis of redox proteins in BAT. Relative protein levels and PRX3 dimer/monomer ratios are presented as fold changes by taking WT as 1.0. n = 2. (E–H) Mitochondrial structures of iBAT by EM. (E) Representative EM images. White boxes denote magnified areas, and arrowheads indicated mitochondria. Quantification of mitochondrial numbers (F), damaged mitochondria percentage (G), and mitochondria with opening outer membrane (H). Ten fields were randomly chosen for each group (n = 3). (I) mtDNA copy number of iBAT (n = 3). Tert was used as a nuclei DNA control. (J and K) mtROS were detected by MitoTracker (green) and MitoSOX (red) in primary brown adipocytes. Arrows indicate MitoSOX⁺ cells. Quantification of RFU is shown in K (n = 3). (L and M) Cytosolic DNA detected by costaining for TOM20 (red) and double-stranded DNA (green) in primary brown adipocytes. Boxes denote magnified areas, and arrows indicate DNA released to the cytoplasm that is quantified in M. (N and O) Cytosolic mtDNA in freshly purified mature brown adipocytes from WT and Trx2^BATKO mice was detected by PCR. (N) Tert expression. (O) Cytosolic mtDNA contents were determined by qPCR with 3 sets of specific primers. Relative mtDNA contents are presented as fold changes by taking WT as 1.0 (n = 5). Quantitative data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Significance was assessed by 1-way ANOVA followed by Tukey’s post hoc test (F–H, N, and O) or 2-tailed Student’s t test (B, C, I, K, and M). Scale bars: 100 μm (A); 0.5 μm (E); 10 μm (J and L). Original magnification for higher magnification images, ×1260 (E and L).