Journal: eLife

Article Title: Metabolomic profiling reveals a differential role for hippocampal glutathione reductase in infantile memory formation

doi: 10.7554/eLife.68590

Figure Lengend Snippet: ( a ) Scheme depicting the glutathione oxidation/reduction cycle and biosynthesis pathway. ( b ) Quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) of reduced (GSH) and oxidized (GSSG) forms of glutathione, of the GSH/GSSG ratio, and of the GSH amino acid precursors glutamate, cysteine, and glycine in hippocampal samples from postnatal day 17 (PN17), PN24, and PN80 (n = 7 rats per group; one experiment). Data are expressed as mean percentage ± s.e.m. of the value in naive adult group (PN80). *p < 0.05; **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( c ) Representative examples and densitometric Western blot analyses of glutathione reductase (GR), glutathione peroxidase (GPx – isozyme 1), glutamate-cysteine ligase (catalytic subunit GCLC, and regulatory subunit GCLM), and glutathione synthetase (GSy), carried out with whole-protein extracts of dHC from PN17 (n = 6), PN24 (n = 4–5), and PN80 (n = 4–5) naive rats; two independent experiments. Actin was used as loading control. Data are expressed as mean percentage ± s.e.m. of the value in naive adult group. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( d ) Mean activity of GR, GPx, GCL, and GSy (expressed as the quantity, in nmol, of substrate transformed per minute and normalized to the total quantity of proteins in each sample, in mg) assayed in dHC protein extracts from PN17 (n = 6), PN24 (n = 6), and PN80 (n = 6) naive rats; two independent experiments. Data are expressed as mean percentage ± s.e.m. of the value in naive adult group. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). See and for numerical values and detailed statistical information. Figure 6—source data 1. Numerical values. Figure 6—source data 2. Detailed statistical information.

Article Snippet: Primary antibodies against the following proteins were obtained from the indicated suppliers: GR (1/2000, Abcam, cat# ab16801), GPx isozyme 1 (1/1000, Abcam, cat# ab22604), GSy (1/1000, Abcam, cat# ab133592), GCL catalytic subunit (1/1000, Novus Biologicals, cat# NBP2-45830), GCL regulatory subunit (1/1000, Abcam, cat# ab126704), NeuN (1/1000, Abcam, cat# ab104225), GFAP (1/2000, Abcam, cat# ab4674).

Techniques: Liquid Chromatography, Tandem Mass Spectroscopy, Western Blot, Control, Activity Assay, Transformation Assay

Journal: eLife

Article Title: Metabolomic profiling reveals a differential role for hippocampal glutathione reductase in infantile memory formation

doi: 10.7554/eLife.68590

Figure Lengend Snippet: ( a ) Quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) of reduced (GSH) and oxidized (GSSG) forms of glutathione and the GSH/GSSG ratio in whole hippocampus from PN17, PN24, and PN80, in the untrained condition and 1 hr after inhibitory avoidance (IA) training (n = 7 rats per group). Data are expressed as mean percentage ± s.e.m. of the value in naive group at each age. ***p < 0.001 (two-way ANOVA followed by Bonferroni’s multiple comparisons test). ( b ) Representative examples and densitometric Western blot analyses of GR, glutathione peroxidase isozyme 1 (GPx), glutamate-cysteine ligase (catalytic subunit GCLC, and regulatory subunit GCLM), and glutathione synthetase (GSy), carried out with whole-protein extracts of dHC from postnatal day 17 naive rats (PN17-N) and rats trained in IA at PN17 and euthanized 1 hr (Tr-1h) or 24 hr (Tr-24h) after training (n = 5–6 rats per group; two independent experiments). Actin was used as the loading control. Data are expressed as mean percentage ± s.e.m. of the value in PN17 naive rats. p > 0.05 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( c ) Mean activity of GR, GPx, GCL, and GSy (expressed as the quantity, in nmol, of substrate transformed per minute and normalized to the total quantity of proteins in each sample, in mg) assayed in dHC protein extracts obtained from rats trained in IA at PN17 and euthanized 15 min, 1 hr, 24 hr, or 7 days after training (Tr-15min, Tr-1h, Tr-24h, and Tr-7d, respectively) (n = 6 rats per group; two independent experiments). To account for developmental differences, two groups of naive (N) rats were used: PN17 and PN24 (n = 6 rats per group). Data are expressed as mean percentage ± s.e.m of the value in PN17 naive rats. *p < 0.05, ***p < 0.001: significance vs. PN17 naive rats (one-way ANOVA followed by Dunnett’s multiple comparisons test); p > 0.05 for the comparison between PN24-N and Tr-7d groups (two-tailed unpaired Student’s t-test). ( d ) GR activity carried out with dHC protein extracts obtained from PN17 naive rats (PN17-N), from rats exposed to an immediate footshock without IA-context exposure (shock-only) and euthanized 15 min later (SO-15min) or from rats trained in IA at PN17 and euthanized 15 min later (Tr-15min) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN17 naive rats. *p < 0.05, **p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( e ) GR activity assayed in dHC protein extracts obtained from PN24 naive rats (PN24-N) and rats trained in IA at PN24 and euthanized 15 min, 1 hr, or 24 hr after training (Tr-15min, Tr-1h, and Tr-24h, respectively) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN24 naive rats. p > 0.05 (one-way ANOVA followed by Dunnett’s multiple comparisons test). ( f ) GR activity assayed in dHC protein extracts obtained from PN80 naive rats (PN80-N) and rats trained in IA at PN80 and euthanized 15 min, 1 hr, or 24 hr after training (Tr-15min, Tr-1h, and Tr-24h, respectively) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN80 naive rats. p > 0.05 (one-way ANOVA followed by Dunnett’s multiple comparisons test). ( g ) Representative examples and densitometric Western blot analyses of NeuN and GFAP carried out with whole-protein extracts of fluorescence-activated cell sorting (FACS)-sorted neurons (NeuN+), astrocytes (GFAP+), and unlabeled counter-selected cells from the dHC of PN17 naive rats (n = 4 x 3 rats per group; two independent experiments). Actin was used as the loading control. NeuN intensity values are expressed as mean percentage ± s.e.m. of the value in neurons (NeuN+) group; GFAP intensity values are expressed as mean percentage ± s.e.m. of the value in astrocytes (GFAP+) group. ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). GR activity carried out with whole-protein extracts of FACS-sorted neurons (NeuN+), astrocytes (GFAP+), and unlabeled cells from the dHC of PN17 naive rats (PN17-N) and of rats trained in IA at PN17 and euthanized 15 min later (Tr-15min) (n = 5 x 3 rats per group; two independent experiments). GR activity is expressed as mean nmol/min/mg protein± s.e.m. *p < 0.05 (two-way ANOVA followed by Bonferroni’s multiple comparisons test). See , for numerical values and detailed statistical information. Figure 7—source data 1. Numerical values. Figure 7—source data 2. Detailed statistical information.

Article Snippet: Primary antibodies against the following proteins were obtained from the indicated suppliers: GR (1/2000, Abcam, cat# ab16801), GPx isozyme 1 (1/1000, Abcam, cat# ab22604), GSy (1/1000, Abcam, cat# ab133592), GCL catalytic subunit (1/1000, Novus Biologicals, cat# NBP2-45830), GCL regulatory subunit (1/1000, Abcam, cat# ab126704), NeuN (1/1000, Abcam, cat# ab104225), GFAP (1/2000, Abcam, cat# ab4674).

Techniques: Liquid Chromatography, Tandem Mass Spectroscopy, Western Blot, Control, Activity Assay, Transformation Assay, Comparison, Two Tailed Test, Fluorescence, FACS

Journal: Molecules

Article Title: Antioxidant and Anti-Inflammatory Activities of a Natural Compound, Shizukahenriol, through Nrf2 Activation

doi: 10.3390/molecules200915989

Figure Lengend Snippet: Figure 3. Induction of antioxidant enzyme gene expression by SZH in BV-2 microglial cells (a) effect of SZH on BV-2 microglial cell viability using CCK-8 cytotoxicity assay for 24 h; (b) expression kinetics of HO-1, a major nrf2-inducing enzyme, by SZH treatment; (c–e) BV-2 microglial cells were harvested after 24 h, the cell lysate were subjected to Western blot; (c) HO-1 (d) GCLM; and (e) GCLC, densitometric analyses were performed and the data were normalized against the internal control β-actin. These data are expressed as fold induction of untreated control ± S.E.M. * p < 0.05, ** p < 0.01 or *** p < 0.005 vs. DMSO only treated control.

Article Snippet: The membranes were incubated overnight with primary antibody against NRF2 (D1Z9C; Cell signaling, Denvers, MA, USA, 1:300), HO-1 (Enzo Life Sciences, Ann Arbor, MI, USA, 1:1000), GCLC (Novus Biologicals, Littleton, CO, USA, 1:1000), GCLM (FL-274; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:1000), iNOS (EPR16635; Abcam, Cambridge, UK, 1:500), NF-κB p65 (C22B4; Cell signaling, Denvers, MA, USA, 1:1000), LAMIN-B1 (D4Q4Z; Cell Signaling, 1:1000), and β-ACTIN (N-21; Santa Cruz Biotechnology, Inc., 1:1000) at 4 °C.

Techniques: Gene Expression, CCK-8 Assay, Cytotoxicity Assay, Expressing, Western Blot, Control

Journal: eLife

Article Title: Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise

doi: 10.7554/elife.41044

Figure Lengend Snippet: Figure 1. Nox4 mediates myocardial Nrf2 activation and is essential in the acute response to exercise. (A) Changes in myocardial Nox4 mRNA and protein levels after acute moderate exercise (Ex) compared to sedentary controls (Sed). *p<0.05, **p<0.01, 2-tailed t-test (n = 4–6/group). (B) Exercise capacity of Nox4-null mice (Nox4KO) and littermate wild-types (WT) measured by maximal running distance and running time. **p<0.01, 2-tailed t-test (n = 5–8/group). (C) Ratio of glutathione/glutathione disulfide (GSH/GSSG) in Nox4KO and WT mouse hearts before (Sed) and after exercise (Ex). n = 6–9/group. (D) Protein levels of 4-hydroxynonenal (4-HNE) adducts in the heart. n = 3–4/group. (E and F) Protein and mRNA levels of major Nrf2 targets. n = 3–6/group *p<0.05, **p<0.01 vs respective sedentary controls (Sed); #p<0.05, ##p<0.01 vs WT/Ex, 1-way ANOVA followed by Tukey post hoc analysis. CAT: catalase, GSTA2: glutathione S-transferase A2, GCLC: glutamate cysteine ligase catalytic subunit, TXNRD1: thioredoxin reductase 1. DOI: https://doi.org/10.7554/eLife.41044.002 The following figure supplements are available for figure 1:

Article Snippet: Antibodies used were: Nrf2, thioredoxin reductase-2, peroxiredoxin III and cadherin (Santa Cruz); Nox4 (Anilkumar et al., 2008); Nox2 (BD Biosciences); p47phox, catalase (Millipore); glutathione S-transferase A2, 4-hydroxynonenal, superoxide dismutase-2 (Abcam); glutamate-cysteine ligase catalytic subunit (Novus biologicals).

Techniques: Activation Assay

Journal: eLife

Article Title: Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise

doi: 10.7554/elife.41044

Figure Lengend Snippet: Figure 2. Cardiac Nox4 deficiency impairs heart function and exercise capacity due to lack of Nrf2 activation. (A) Expression of Nrf2-related genes in hearts of cardiomyocyte-specific Nox4KO (csNox4KO) and littermate controls after exercise (Ex). n = 3–4/group. **p<0.01 vs respective sedentary controls (Sed); #p<0.05, ##p<0.01 vs Control/ Ex. (B) Maximal exercise distance and time. n = 3–5/group. *p<0.05 vs Control/Ex; #p<0.05, ##p<0.01 vs csNox4KO/Ex, 1-way ANOVA followed by Tukey post hoc analysis. (C) Fractional shortening (FS) evaluated by conscious echocardiography in control and csNox4KO mice immediately after the exercise capacity test. Some animals were treated with sulforaphane (SFN) prior to exercise. n = 4–12/group. **p<0.01 vs Sed; ##p<0.01 vs Control/Ex, 2-way ANOVA followed by Tukey post hoc analysis. Cat: catalase, Gsta2: glutathione S-transferase A2, Gclc: glutamate cysteine ligase catalytic subunit, Txnrd1: thioredoxin reductase 1. DOI: https://doi.org/10.7554/eLife.41044.006 The following figure supplement is available for figure 2:

Article Snippet: Antibodies used were: Nrf2, thioredoxin reductase-2, peroxiredoxin III and cadherin (Santa Cruz); Nox4 (Anilkumar et al., 2008); Nox2 (BD Biosciences); p47phox, catalase (Millipore); glutathione S-transferase A2, 4-hydroxynonenal, superoxide dismutase-2 (Abcam); glutamate-cysteine ligase catalytic subunit (Novus biologicals).

Techniques: Activation Assay, Expressing, Control

Journal: Orthopaedic Journal of Sports Medicine

Article Title: Clinical Outcome 3 Years After Autologous Chondrocyte Implantation Does Not Correlate With the Expression of a Predefined Gene Marker Set in Chondrocytes Prior to Implantation but Is Associated With Critical Signaling Pathways

doi: 10.1177/2325967114550781

Figure Lengend Snippet: Repressed Genes in the Success Group

Article Snippet: The genes analyzed with qPCR were selected from the predefined marker set and from genes annotated with enriched biological process terms in the gene ontology (GO) analysis (assay number in parentheses): COL2A1 (Hs00156568_m1), BMP2 (Hs00154192_m1), FGFR3 (Hs00179829_m1), ALK1 (Hs00163543_m1), ACAN (Hs00153936_m1), CD44 (Hs01075862_m1), PTPRD (Hs00369913_m1), PTPRF (Hs00892965_m1), ST6GAL2 (Hs00383641_m1), GCLC (Hs00155249_m1), PRLR (Hs01061477_m1), GRIK2 (Hs00222637_m1), LYST (Hs00915897_m1), AGT (Hs01586213_m1), FAIM2 (Hs00392342_m1), PHLDA1 (Hs00378285_g1).

Techniques: Binding Assay, Variant Assay, Sequencing