Fig 1: Protein immunoblot analysis. Immunoblotting of the proteins involved in mitochondrial and metabolic functions was performed in the hippocampi from the control, depression-susceptible, anxiety-susceptible, and insusceptible groups.Por, Idh2, Esd, Glo1, G6pdx, Aldh2, Dld, Dlat, Ogdhl, Anxal, Tpp2, Sdha, Prnp, Pkm, and Prdx6 were detected with their respective antibodies. Each blot corresponded to the five rats used in the analysis, and Coomassie blue staining was used as the loading control. Dep-Sus depression-Susceptible, Anx-Sus Anxiety-susceptible, Insus Insusceptible, Cont Control; n = 5, *p < 0.05, **p < 0.01
Fig 2: A. Representative immunoblots for iNOS, DLAT, DLST, DBT, and lipoic moiety in whole cell lysates of unstimulated C2C12 myotubes or myotubes stimulated with TNFa and IFN? for 48 hours, with or without treatment with 1400W (200 µM). The experiment was repeated four times. To compare the changes in functional lipoic arm upon stimulation, the relative ratio of lipoic band to its corresponding total E2 subunit band (DLAT for the lipoic band at ~70kDa, and DLST or DBT for the lipoic band at ~50kDa) was quantified and normalized to unstimulated, untreated condition, as graphed in the corresponding figure. Each dot represent result for each independent experiments B. BCKDC activity, as measured by isovaleryl-CoA production from aKIC over time in crude mitochondria isolation from C2C12 myotubes treated with or without 1400W (200 µM) and with or without TNFa and IFN? stimulation. C. Representative immunoblot for myosin heavy chain (MHC) and beta-actin over C2C12 differentiation time course from wild type and Nos2-/- whole cell lysate when cells were in growth media (GM) and after the first (D1) and fifth day (D5) in differentiation media. This experiment was repeated three times. MHC expression was quantified and normalized to loading control (beta-actin) in each experiment. Relative expression was graphed as relative level compared to wild type D5 condition. Each dot represents an independent experiment. D. The same as Fig. 2A but with genetic Nos2 knock out instead of 1400W treatment. E. Relative abundance of aKIC (6-labeled from U-[13C]-L-leucine) in stimulated (TNFa and IFN? for 48 hours) or unstimulated wild-type or Nos2-/- C2C12 myotubes. F. The fraction of M+2 labeled acetyl-CoA after 48 hours labeling with U-[13C]-L-leucine in stimulated or unstimulated wild-type or Nos2-/- C2C12 myotubes. A, C, and D. For immunoblot quantification, statistical analysis was performed with unpaired student’s t-test with p-value of significant changes indicated on graph. ns indicate no significant change (p>0.05). B. Plots represent mean ± standard deviation, n = 3. Statistical analysis was performed with unpaired student’s t-test with p-value significant indicated on graph. E and F. All bars and error bars represent mean ± standard deviation, n = 3. Statistical analysis for significance was performed with one-way ANOVA followed by a post-hoc Tukey’s test. Columns with different letters indicate a statistical significance of p<0.05.
Fig 3: Western blot analysis in patient lymphoblasts (A–C) and fibroblasts (D).(A) Western blot analysis to assess the expression level of one subunit of each of the five different oxidative phosphorylation complexes in the patient lymphoblast sample (P) compared to three control lymphoblast lines from healthy individuals (C1–3). Protein content of NDUFB8 and COXII, which are subunits of complexes I and IV, respectively, are decreased in the patient compared to healthy controls. (B) Western blot analysis to assess malonyl-CoA-acyl carrier protein transacylase (MCAT) levels reveals decreased expression of MCAT in patient lymphoblasts (P) compared to three controls (C1–3). (C) Western blot analysis to assess lipoylation with an anti-lipoic acid antibody in patient lymphoblasts (P) compared to controls (C1–3) reveals normal lipoylation of the PDH and OGDH E2 components (DLAT and DLST, respectively) in the patient sample. (D) Western blot analysis to assess the expression level of one subunit of each of the five different oxidative phosphorylation complexes in the patient fibroblast sample (P) compared to five fibroblast controls (C1–5). Protein content of NDUFB8, COXII, and SDHB, which are subunits of complexes I, IV, and II, respectively, are decreased in the patient compared to healthy controls. Figure 2—source data 1.Uncropped immunoblots for Figure 2. Figure 2—source data 2.Unlabeled immunoblots for Figure 2.
Fig 4: A.B. Relative overall PDHC enzymatic activity (A) or DLD activity (B) measured by spectrometric assay after purified porcine PDHC was incubated with indicated combination of PAPA-NONOate (1mM), pyruvate (1mM) and CoA (100 µM). Statistical analysis was performed with student’s t-test, with all significant differences (p<0.05) indicated on figure with the corresponding p-value. All bars and error bars represent mean ± standard deviation, n = 3.C. in-gel assay after purified porcine PDHC was incubated with indicated combination of PAPA-NONOate (1mM), pyruvate (1mM) and CoA (100 µM). DLD activity is indicated by purple dye intensity at the molecular weight of DLD dimer (~146 kDa) in native gel. Samples loaded on gel in duplicates. D. Immunoblot for total DLD level in the same samples as (C). E. Schematic showing the function of MECR and that MECR knockout HAP1 cells lack lipoic arm on their a-ketoacid dehydrogenase complexes. F. Immunoblot for lipoic moiety, DLAT and DLD in wild type and MECR-/- HAP1 cells with or without 250 µM DETA-NONOate treatment. G. Relative enzymatic activity of PDHC in wild type or MECR-/- HAP1 cells with or without 250 µ DETA-NONOate treatment. H. Relative enzymatic activity of DLD measured by spectrometric assay in DETA-NONOate (250 µM) treated wildtype or MECR-/- cells compared to untreated condition. G.H. Statistical analysis was performed with unpaired student’s t-test with p value reported. Bars and error bars represent mean ± standard deviation, n = 3.
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