Figure 5.

Trx2 deficiency increases mitochondrial ROS generation and NF-κB activation. (A and B) Immunoblot analysis of proteins involved in mitochondrial dynamics and mitophagy in eWAT of WT and Trx2^ADKO mice at 14 and 24 wk (n = 3). β-Actin was used as a loading control. (C) Representative micrographs of eWAT sections showing ROS levels, as assessed by the mitochondrial-specific ROS probe mitoSOX, and quantification of fluorescence intensity in WT and Trx2^ADKO mice at 14 and 24 wk (n = 6). AFU, arbitrary fluorescence unit. (D) Representative Western blot showing protein levels of Trx2, Prx3, Glut4, HSL, and PPARγ in eWAT from WT and Trx2^ADKO mice at 14 and 24 wk. (E) Immunoblot analysis of IKK–NF-κB signaling molecules (phospho-IKKα/β, IKKβ, phospho-P65, P65, phospho-IκB, IκB, phospho-AKT, and AKT) in eWAT from WT and Trx2^ADKO mice at 14 and 24 wk. Protein levels (A, B, D, and E) were quantified and presented as fold changes by taking WT as 1.0. n = 3 mice for each group. (F) Coimmunostaining of phospho-P65 (green) and adipocyte marker FABP4 (red) in eWAT sections from 14-wk-old male WT and Trx2^ADKO mice (n = 6). White arrowheads indicate phospho-p65 in the nucleus. (G) Coimmunostaining of P65 (green) and macrophage marker F4/80 (red) in eWAT sections from 14-wk-old male WT and Trx2^ADKO mice.(H and I)Quantification of TNF-α and IL-6 in eWAT and serum of WT and Trx2^ADKO mice at 14 and 24 wk (n = 8). Data are presented as mean ± SEM. **, P < 0.01; ***, P < 0.001 for the indicated comparisons. Significance was assessed by two-tailed Student’s t test. Scale bars, 50 µm (C); 20 µm (F and G). w, weeks.