Fig 2: NNT loss does not compromise the mitochondrial TXN antioxidant system. (A) Immunoblot analysis of NNT and actin (loading control) expression in NSCLC cells 3 d after infection with scramble or shNNT lentivirus. (B) Relative cell density of NSCLC cells 4 or 7 d after infection with scramble or shNNT lentivirus (one-way ANOVA). (C) Relative viability of NSCLC cells 4 or 7 d after infection with scramble or shNNT lentivirus (one-way ANOVA). (D) Schematic representation of the canonical function of NNT, where NNT supplies NADPH to maintain mitochondrial antioxidant capacity. TXN: oxidized (TXNo), reduced (TXNr). (E) NADPH:NADP+ ratio in NSCLC cells following NNT knockdown, relative to scramble infected control cells (Student’s t test). (F) Fluorescence of the mitochondrial H2O2 sensitive dye, MitoPY1, in NSCLC cells following NNT knockdown relative to scramble infected control cells (one-way ANOVA). (G) Redox immunoblot analysis of HSP90 (loading control) and the oxidation state of PRDX3 in parental NSCLC cells following 4 h treatment with DMSO, 2 µM, or 4 µM auranofin or 4 d after infection with scramble or shNNT lentivirus. Data are representative of one experiment of three experimental replicates. For B–F, data are represented as mean ± SD of three technical replicates. n.s., not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 3: NNT loss disrupts fatty acid metabolism. (A) Heat map representation of the relative abundances of long chain fatty acyl-carnitines in extracts of NSCLC cells following NNT knockdown. (B) Measures of OCR coupled to the oxidation of exogenous palmitate-BSA in NSCLC cells subject to NNT knockdown (Student’s t test). (C) Heat map representation of the relative abundances of saturated (decanoate, lauric acid, palmitate, stearate) and unsaturated (palmitoleic acid, elaidic acid, linoleate) fatty acids in extracts of NSCLC cells following NNT knockdown. (D) Relative BODIPY-FL C16 fluorescence of scramble or shNNT-infected NSCLC cells following a 30-min incubation with the fluorescent palmitate analogue (Student’s t test). (E and F) Viability of NSCLC cells subject to scramble or shNNT lentiviral infection following 24 h treatment with (E) 200 µM of palmitate or (F) 400 µM of oleate (Student’s t test). Cell viability was determined relative to BSA treated controls. (G) Viability of NSCLC cells subject to scramble or shNNT lentiviral infection following a 48-h incubation in cell culture media with or without serum lipids. Cell viability was determined relative to scramble controls treated with lipid replete media. (H) NADPH:NADP+ ratio in NSCLC cells subject to scramble or shNNT lentiviral infection following a 24-h incubation in cell culture media with or without serum lipids. Ratio normalized to scramble controls incubated in lipid replete conditions (two-way ANOVA). For A and C, data are represented as the mean fold increase relative to scramble controls of three biological replicates. For B, D, and E–H, data are representative of one experiment of three experimental replicates. For B, D, and E–H, data are represented as mean ± SD of three technical replicates. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 4: NNT loss does not sensitize NSCLC cells to exogenous oxidants. (A) Immunoblot analysis of NNT and HSP90 (loading control) expression in a panel of human NSCLC cells. (B) Plots of cell proliferation over 7 d for NSCLC cells subject to scramble or shNNT lentiviral infection. (C) Fluorescence of the mitochondrial •O2- sensitive dye, MitoSOX Red, in NSCLC cells following NNT knockdown, relative to scramble-infected control cells (one-way ANOVA). (D) Immunoblot analysis of NNT, TRXR2, TXN2, and HSP90 (loading control) in lysates of NSCLC cells 4 d after lentiviral infection. (E–H) Viability of NSCLC cells subject to scramble or shNNT lentiviral infection following 24 h treatment with (E) 1 µM auranofin or 15 µM (F) tbHP, (G) CHP, or (H) menadione (Student’s t test). Cell viability was determined relative to vehicle treated controls. Data are representative of one experiment of three experimental replicates. For B, C, and E–H, data are represented as mean ± SD of three technical replicates. n.s., not significant; *, P < 0.05; ***, P < 0.001.

Fig 5: The effect of NNT loss on ACO2 is independent of changes in protein expression. (A) Average coupling efficiency of A549 and PC9 cells following infection with either scramble or shNNT lentivirus (Student’s t test). (B) Metabolic phenotyping of NSCLC cells subject to 10 mM glucose and 1 mM glutamine before (open circles) and after cotreatment (closed circles) with oligomycin (1 µM) and FCCP (0.5 µM for H441, H1299, H2009, PC9 cells; 1 µM for A549). Dotted lines are representative of metabolic potential under stress conditions. (C) Ratio of basal oxygen consumption (OCR) and extracellular acidification (ECAR) in NSCLC cells subject to 10 mM glucose and 1 mM glutamine. (D) ECAR values of NSCLC cells subject to 10 mM glucose following treatment with 1 µM oligomycin. (E) Immunoblot analysis of ACO2 and actin (loading control) expression in NSCLC cells subject to NNT knockdown. (F) Average complex I–III activity following stimulation with 1 mM malate and either 10 mM pyruvate or 10 mM glutamate in H2009 cells subject to NNT knockdown (one-way ANOVA). (G) Average complex II–III activity in H2009 cells subject to NNT knockdown following stimulation with 10 mM succinate in the presence of 1 mM malate and either 10 mM pyruvate or 10 mM glutamate (one-way ANOVA). (H) Immunoblot analysis of Nnt, Aco2, and Hsp90 (loading control) expression in lung tumors collected from LSL-KrasG12D/+; Trp53flox/flox; Nnt+/+ (n = 6), LSL-KrasG12D/+; Trp53flox/flox; Nnt?ex7-11/+ (n = 6), and LSL-KrasG12D/+; Trp53flox/flox; Nnt?ex7-11/?ex7-11 (n = 6) mice. Data are representative of one experiment of three experimental replicates. For A–D, F, and G, data are represented as mean ± SD of at least three technical replicates. n.s., not significant; ***, P < 0.001.