Fig 1: NNT loss does not disrupt Fe-S cluster biosynthesis. (A) Average complex I–III activity following stimulation with 10 mM pyruvate and 1 mM malate in H2009 and PC9 cells subject to NNT, NFS1, or ISCU knockdown (one-way ANOVA). (B) Average complex II–III activity following stimulation with 10 mM pyruvate and 1 mM malate in H2009 and PC9 cells subject to NNT, NFS1, or ISCU knockdown (one-way ANOVA). (C) Average ACO2 activity in mitochondrial lysates of NSCLC cells following NNT, NFS1, or ISCU knockdown (one-way ANOVA). (D) Relative abundance of TCA cycle intermediates in extracts of NSCLC cells subject to NNT or ISCU knockdown (one-way ANOVA). (E) Immunoblot analysis of NNT, lipoic acid, PDH-E2, DLST, NFS1, ISCU, and actin (loading control) expression in PC9 cells following infection with (1) scramble, (2) shNNT-507, (3) shNNT-512, (4) shNFS1_1, (5) shNFS1_2, or (6) ISCU lentivirus. For A–C, data are representative of one experiment of three experimental replicates. For A–C, data are represented as mean ± SD of at least three technical replicates. For D, data are represented as mean ± SD of three biological replicates. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 2: Palmitate lowers nicotinamide nucleotide transhydrogenase (NNT) protein expression: Cellular (A) NNT, (B) isocitrate dehydrogenase 2 (IDH2), (C) malic enzyme 2 (ME2), or (D) the glutamate cysteine ligase catalytic subunit were measured relative to the β actin loading control on Western blots to assess the redox regulators in PBMCs stimulated with αCD2/αCD28 in the presence or absence of palmitate (pal) and oleate (ole) per Figure 1. In panel A, N = 7–8. For Panels B,C&D, N = 5. Each N represents cells from one subject. * p < 0.05 vs. control (con).
Fig 3: NNT knockdown promotes ROS and Th17 cytokine production: (A) NNT protein measured following knockdown with scrambled (scr) or NNT-targeted siRNA. Amounts of cellular (B) ROS (DCFDA), (C) NADPH (measured using a commercial kit), (D) IL-17A/F (in ELISA) and (E) IL-23 (in ELISA). (F) IL-17A/F quantified after stimulation in the presence of the indicated additives: palmitate (pal), oleate (ole), MitoTempo (mito) (10 µM) or N-acetyl cysteine (NAC, 20 µM). A,B,C,F N = 5, D&E N = 7–8 in each treatment. Each N represents cells from one subject. * p < 0.05 vs scr siRNA treated cells (A–E) or pal in F.
Fig 4: Chest blast exposure alters the expression of mitochondrial proteins in the brain. (A) Western blot images of differentially expressed proteins after chest blast exposure. The expression levels of (B) Th, (C) Nnt, and (D) Clic1 with normalized to internal control GAPDH. All experiments were repeated at least three times. All data were expressed as mean ± SEM (n = 5) and analyzed by a one-way ANOVA, followed by Tukey’s test for multiple comparisons. Differences were considered statistically significant at p < 0.05 for all analyses. *p < 0.05 vs. control group.
Fig 5: NNT loss compromises mitochondrial oxidative capacity. (A) Plots of OCR in A549 and PC9 cells subject to NNT knockdown. Cells were supplemented with 10 mM glucose and 1 mM glutamine and then sequentially challenged with 1 µM oligomycin (Olig), 1 µM (A549) or 0.5 µM (PC9) of FCCP, and 1 µM each of antimycin A (A) and rotenone (R). (B) Average maximal respiratory capacity of A549 and PC9 cells following infection with either scramble or shNNT lentivirus (Student’s t test). (C) Average complex I–III activity following stimulation with 10 mM pyruvate and 1 mM malate in NSCLC cells subject to NNT knockdown (one-way ANOVA). (D) Average complex II–III activity following stimulation with 10 mM succinate in NSCLC cells subject to NNT knockdown (one-way ANOVA). (E) Average ACO2 activity in mitochondrial lysates of NSCLC cells following NNT knockdown (one-way ANOVA). (F) Average ACO2 activity in mitochondrial lysates of lung tumors collected from LSL-KrasG12D/+; Trp53flox/flox; Nnt+/+ (n = 13 tumors from five mice), LSL-KrasG12D/+; Trp53flox/flox; Nnt?ex7-11/+ (n = 20 tumors from five mice), and LSL-KrasG12D/+; Trp53flox/flox; Nnt?ex7-11/?ex7-11 (n = 18 tumors from four mice) mice (one-way ANOVA). For A–E, data are representative of one experiment of three experimental replicates. For A–E, data are represented as mean ± SD of at least three technical replicates. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. pmoles, picomoles; RFU, relative fluorescence units.
Supplier Page from Abcam for Anti-NNT antibody [8B4BB10]