Fig 1: Redox parameters of GPx2 and GPx1 kd cells. Scr, GPx1 and GPx2 kd HT-29 cells were supplemented with 50 nM sodium selenite for 72 h. Cells were harvested and GPx activity was measured using different substrates (H2O2, HPODE, HPETE, and TBHP) with a final concentration of 50 µM (A). Numbers within the bars represent percentages in comparison to the respective scr set as 100%. For the DHR123 assay, cells were incubated with 5 µM DHR for 45 min followed by 1 h treatment with or without 5 or 10 ng/mL IL-1ß (B) or 1 mM H2O2, 50 µM HPODE, HPETE, or TBHP (C). Values of the DHR assay were normalized to viable cells analyzed by neutral red assay. NOX1 protein levels were analyzed by Western Blot 4 h after 1 ng/mL IL-1ß stimulation and normalized to Coomassie staining. Data are given as means + SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 vs. scr and ##p < 0.01; ###p < 0.001 vs. shGPx2#1 analyzed by two-way ANOVA with Bonferroni's post-test.
Fig 2: TRIM28 negatively regulates GPX1 stability via ubiquitination. (A) H/R-induced AC16 cells were transduced with lentivirus siTRIM28 or siNC and (B) AC16 cells were transduced with lentivirus oeTRIM28. After 24 h, the mRNA and protein levels of GPX1 were measured by reverse transcription-quantitative PCR and western blotting, respectively. (C) Interaction of TRIM28 and GPX1 was detected by co-immunoprecipitation. (D) GPX1 antibody was used to pull down immunocomplex. Ubiquitin antibody was used for subsequent western blotting. ####P<0.0001 vs. Control. TRIM, tripartite interaction motif; GPX, glutathione peroxidase; H/R, hypoxia/reoxygenation; si, small interfering RNA; NC, negative control; oe, overexpression.
Fig 3: ROS detoxifying enzymes and MDA levels are not altered in L6 myotubes following DDE exposure. Levels of (A) Grx2, (B) SOD2, (C) GPx1, and (D) catalase measured in L6 myotubes exposed for 24 h to 0, 1, 10, 100, 1000, and 10,000 nM of DDE. Alpha tubulin was used as a loading control. Top panels: representative Western blots, bottom panels: quantification by density analysis. n = 3 independent experiments. Mean ± SEM. (E) Intracellular MDA concentration (nmol MDA/mg of protein) measured in L6 myotubes exposed for 24 h to 0, 1, 10, 100, 1000, and 10,000 nM of DDE. n = 4 independent experiments for all but 10,000 nM DDE (n = 3). Mean ± SEM.
Fig 4: Long-term tBHQ-stimulated changes in the protein expression of antioxidant responsive genes. Different lines of WT (A), Nrf1a-/- (B), Nrf2-/-?TA (C), and caNrf2?N (D) were treated with 50 µM tBHQ or not for 0 to 24 h, before basal and stimulated abundances of those antioxidant cytoprotective proteins, e.g., GCLC (a1, b1, c1, d1), GCLM (a2, b2, c2, d2), GSR (a3, b3, c3, d3), GPX1 (a4, b4, c4, d4), and TALDO (a5, b5, c5, d5), were determined by western blotting with the indicated antibodies. The intensity of relevant immunoblots representing different protein expression levels was also quantified by the Quantity One 4.5.2 software. The resulting data were then shown graphically (in right panels), after being calculated by a formula of Ln([A]t/[A]0), in which [A]t indicated a fold change (mean ± SD) in each of those examined protein expression levels at different times relative to the corresponding controls measured at 0 h (i.e., [A]0), which were representative of at least three independent experiments.
Fig 5: The expression of GPX-1 of mouse skin samples of ACF-treated group and control group. (A) The expression of antioxidant enzyme GPX-1 was detected in both the ACF-treated and control groups at each time point. (B) Quantitative reverse transcription PCR analysis of the GPX-1 expression in the dermis of both groups. The higher expression of GPX-1 was detected in the ACF-treated group than that in control groups at week 1. P (week 1) = 0.003, F (week 1) = 0.105; P (week 2) = 0.303, F (week 2) = 0.303; P (week 4) = 0.145, F (week 4) = 0.228. Scale bar = 100 μm.
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