Trx2 deficiency promotes mitophagy through NF-κB–dependent p62/SQSTM1 accumulation and mitochondrial recruitment. (A) Representative transmission electron micrographs showing typical mitophagy in differentiated adipocytes of Trx2^ADKO mice on day 8 (n = 3 mice/group). Squares correspond to the magnified areas. Arrows indicate mitochondria. Scale bars, 1 µm. (B) Percentage of damaged mitochondria in 10 random fields (n = 20 cells/group). (C) Immunoblot analysis of Trx2, mitophagy receptors p62 and NBR1, and autophagy markers LC3 and Beclin-1 in differentiated adipocytes of WT and Trx2^ADKO mice during differentiation. (D) Subcellular distribution of p62, LC3, and Beclin-1 in differentiated adipocytes of WT and Trx2^ADKO mice at day 4. Voltage-dependent anion channel was used as a mitochondrial control. (E) Immunoblot analysis of mitophagy-related proteins in mitochondrial fraction of differentiated adipocytes of WT and Trx2^ADKO mice at day 4 of differentiation. Inner membrane protein TOMM20 was used as a control. (F) Immunoblot analysis of NF-κB signaling proteins from differentiated adipocytes (day 4) of WT and Trx2^ADKO mice in the absence or presence of mito-TEMPO. (G) Representative immunoblot analysis of mitochondrial dynamics and mitophagy-related proteins from day differentiated adipocytes of WT and Trx2^ADKO mice in the absence or presence of mito-TEMPO. (H) Representative immunoblot of NF-κB signaling proteins and mitophagy-related proteins with and without IKK inhibitor BMS-345541 in differentiated adipocytes (day 4) of WT and Trx2^ADKO mice. Protein levels (C–H) were quantified and presented as fold changes by taking untreated WT as 1.0. n = 3 mice for each group. (I) Coimmunoprecipitation (IP) was performed using anti-IgG or anti-p62 and immunoblotting (IB) with anti-ubiquitin antibody (Ub) in differentiated adipocytes of WT and Trx2^ADKO mice at day 4. The experiment was repeated three times. (J) Confocal fluorescence images showing colocalization of p62 (green) and polyubiquitin (poly-Ub, purple) with mitochondria (TOMM20, red) in adipocytes of WT and Trx2^ADKO mice during differentiation. Images are representative of three independent experiments. Scale bars, 10 µm. Quantitative data represent the mean ± SEM. ***, P < 0.001 versus WT (two-tailed Student’s t test).