Fig 2: Adropin expression in the mouse brain.A Expression profiling in mouse brain structures by age show a peak at postnatal day 32 (P32). B, C Adropin immunoreactivity in brain lysates of AdrTG mice compared to wild type (WT) littermates at 4 and 18 months of age. WT mice show a decline in adropin protein levels at 18 months that is not observed in AdrTG. HSP90 was used as a loading control. D Profiling of Enho expression in isolated cells from the mouse brain. E The top panel is a schematic of the approach used to generate an ENHO-Cre reporter mouse. The lower panels (F–Q) are representative photomicrographs from hippocampal sections (F–K) and high magnification photomicrographs of vessels in the cortex (L–Q). Scale bars (10 µM) are shown in the bottom left in (L–Q).

Fig 3: NNT knockdown does not induce cytosolic oxidative stress. (A) Fluorescence of the cytosolic ROS-sensitive dye, CellROX Green, in NSCLC cells following NNT knockdown, relative to scramble-infected control cells (one-way ANOVA). (B) Redox immunoblot analysis of HSP90 (loading control) and the oxidation state of PRDX1 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. (C) Immunoblot analysis of protein expression in lysates of NSCLC 4 d after lentiviral infection; HSP90 (loading control), NNT, NRF2 and its downstream targets TRXR1, NQO1, TXN1. (D) Relative abundance of reduced GSH in extracts of NSCLC cells subject to NNT knockdown (one-way ANOVA). (E) Relative abundance of oxidized glutathione (GSSG) in extracts of NSCLC cells subject to NNT knockdown (one-way ANOVA). (F and G) Viability of NSCLC cells subject to scramble or shNNT lentiviral infection following 24 h treatment with 100 µM (F) BSO or (G) 6-aminonicotinamide (6-AN; Student’s t test). (H) Redox immunoblot analysis of the oxidation state of TXN1 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. Control samples derivatized in either iodoacetic acid (IAA) or iodoacetamide (IAM) were included to denote oxidized and reduced TXN1 signals, respectively. For A–C and F–H, data are representative of one experiment of three experimental replicates. For D and E, data are represented as the mean fold change relative to scramble controls of three biological replicates. For A and D–G, data are represented as mean ± SD of three technical or biological replicates. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4: 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 5: (A) Western blot showing that BMP2, annexin A6 and annexin A2 carriage to be higher in MVs from senescent than from young HUVEC. Equal protein loading was confirmed using a HSP90 antibody. Total protein expression was obtained via densitometric analysis and expressed as the ratio protein/HSP90 in arbitrary units. n=3 pools; *p<0.05; **p<0.01. (B) Representative BMP2, annexin A6 and annexin A2 western blot of young and senescent HUVEC pools. Equal protein loading was confirmed probing with GAPDH or ß-Actin. The graphs present densitometric band analysis normalized to GAPDH or ß-Actin in arbitrary units. n=3 pools.