Journal: Pharmaceutics

Article Title: Colon-Targeted Poly(ADP-ribose) Polymerase Inhibitors Synergize Therapeutic Effects of Mesalazine Against Rat Colitis Induced by 2,4-Dinitrobenzenesulfonic Acid.

doi: 10.3390/pharmaceutics16121546

Figure Lengend Snippet: Figure 3. AQSA-Glu potentiates the anticolitic activity of 5-AIQ. Three days after colitis induction by DNBS, 5-AIQ (5 mg/kg) and AQSA-Glu [7.5 mg/kg, equivalent to 2.5 mg/kg of 5-AIQ (L) and 15 mg/kg, equivalent to 5 mg/kg of 5-AIQ (H)] were administered orally to rats once per day, and the rats were euthanized 24 h after the sixth treatment. (A) Left panel: photos of the distal colons of rats in which serosal and luminal sides are shown separately. Right panel: overall colonic damage was scored for each group and presented as colonic damage score (CDS). * α < 0.05 vs. DNBS control. (B) H & E staining was performed with the colonic tissue sections of rats subjected to various treatments. Upper panel: representative images of 100× magnification. Lower panel: representative images of 200× magnification for the dotted boxes in the upper panel. In the inflamed distal colons (4.0 cm), (C) myeloperoxidase (MPO) activity was measured in addition to determining the levels of (D) CINC-3 and (E) iNOS and COX-2 using an Elisa kit and Western blotting. A loading control (α-Tubulin) was used for Western blot analysis of COX-2 and iNOS. NM: not measurable. The data are represented as mean ± SD (n = 5). * p < 0.05 vs. DNBS control # p < 0.05.

Article Snippet: Nrf2, HO-1, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) proteins were detected using the following antibodies: anti-Nrf2 (sc-365949, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO-1 (sc-136961, Santa Cruz Biotechnology), anti-COX-2 (sc-365374, Santa Cruz Biotechnology), anti-iNOS (NOS-2) antibody (sc-7271, Santa Cruz Biotechnology), and secondary antibodies corresponding to the primary antibodies (Santa Cruz Biotechnology).

Techniques: Activity Assay, Control, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Pharmaceutics

Article Title: Colon-Targeted Poly(ADP-ribose) Polymerase Inhibitors Synergize Therapeutic Effects of Mesalazine Against Rat Colitis Induced by 2,4-Dinitrobenzenesulfonic Acid.

doi: 10.3390/pharmaceutics16121546

Figure Lengend Snippet: Figure 4. Colon-targeted PARP inhibitors synergize the anticolitic effects of mesalazine. (A) RAW264.7 cells pretreated with 5-ASA (20 mM), 3-AB (1 mM), and 5-AIQ (10 µM) for 1 h were challenged with LPS for 24 h. The levels of iNOS and COX-2 proteins were analyzed using West- ern blotting. (B) SSZ (50 mg/kg) and ABSA (36 mg/kg, equimolar to 50 mg/kg of SSZ) suspended in PBS (1 mL) were administered orally to rats. The rats were killed 2, 4, and 8 h after oral administration. The concentrations of 5-ASA in the cecum were analyzed using HPLC. (C) Three days after colitis induction by DNBS, SSZ (50 mg/kg), AQSA-Glu (15 mg/kg), a mixture of AQSA-Glu (15 mg/kg) + olsalazine (OSZ, 19 mg/kg, half-equimolar to 50 mg/kg of SSZ), and ABSA (36 mg/kg, equimolar to 50 mg/kg of SSZ) were administered orally to rats once per day, and the rats were euthanized 24 h after the sixth treatment. (C) Left panel: photos of the distal colons of rats where serosal and luminal sides are shown separately. Right panel: overall colonic damage was scored for each group and presented as colonic damage score (CDS). * α < 0.05 vs. DNBS control. (D) H & E staining was performed with the colonic tissue sections of rats subjected to various treatments. Upper panel: rep- resentative images of 100× magnification. Lower panel: representative images of 200× magnification for the dotted boxes in the upper panel. In the inflamed distal colons (4.0 cm), (E) myeloperoxidase (MPO) activity was measured in addition to determining the levels of (F) CINC-3 and (G) iNOS and COX-2 using an Elisa kit and Western blotting. A loading control (α-Tubulin) was used for Western blot analysis of COX-2 and iNOS. NM: not measurable. The data are represented as mean ± SD (n = 5). * p < 0.05 vs. DNBS control # p < 0.05.

Article Snippet: Nrf2, HO-1, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) proteins were detected using the following antibodies: anti-Nrf2 (sc-365949, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO-1 (sc-136961, Santa Cruz Biotechnology), anti-COX-2 (sc-365374, Santa Cruz Biotechnology), anti-iNOS (NOS-2) antibody (sc-7271, Santa Cruz Biotechnology), and secondary antibodies corresponding to the primary antibodies (Santa Cruz Biotechnology).

Techniques: Control, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

doi: 10.1073/pnas.1417566112

Figure Lengend Snippet: Preternaturally long-lived naked mole-rats have higher Nrf2-signaling activity than short-lived mice. (A) Liver tissue of naked mole-rats have higher Nrf2–ARE-binding (n = 4) activity than that of mice (P = 0.0001). (B and C) Nrf2 total protein levels are higher (B; n = 6; P = 0.0245) and Keap1 levels lower (C; P = 0.0037) in naked mole-rats. (D and E) Quantitative PCR similarly shows that Nrf2 transcript levels are markedly higher (D; P = 0.0195) and Keap1 levels strikingly lower (E; P = 0.0001) in the longer-lived species. These data indicate that the higher Nrf2:ARE binding activity is due to increased Nrf2 protein availability as a result of low levels of Keap1 and concomitant decreased Nrf2 ubiquitination and proteasomal degradation. Error bars represent SEM.

Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

Techniques: Activity Assay, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

doi: 10.1073/pnas.1417566112

Figure Lengend Snippet: Transcript expression of downstream molecules regulated by Nrf2 is significantly higher in naked mole-rats than in mice. Naked mole-rats have higher levels of several gene transcripts that are dominantly regulated by Nrf2 (n = 5 or 6). (A) These include Hmox1 (Left; P = 0.0376), Gsta1 (Center; P = 0.0336), and Nqo1 (Right; P = 0.0200). (B and C) Elevated Nrf2-signaling activity in the naked mole-rat was further confirmed via higher total GST (B; P = 0.0001) and NQO1 activities (C; P = 0.0002) than observed in mice. Error bars represent SEM.

Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

Techniques: Expressing, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

doi: 10.1073/pnas.1417566112

Figure Lengend Snippet: Nrf2–ARE binding activity is significantly correlated with the lifespan of rodent species. (A) Ten different rodent species with varying MLSPs were used to asses basal Nrf2 signaling activities. (B) Liver Nrf2–ARE binding activity was positively correlated with MLSP in the 10 rodent species (n = 4 per species; P = 0.0285), whose MLSP varied between 4 and 31 y and whose body weights ranged from 20 g to almost 1 kg. (C) Nrf2 signaling did not correlate with bodyweight (P = 0.6082). Error bars represent SEM.

Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

Techniques: Binding Assay, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

doi: 10.1073/pnas.1417566112

Figure Lengend Snippet: Multiple proteins that regulate Nrf2-signaling activity correlated with MLSP in rodents

Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

Techniques: Activity Assay, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

doi: 10.1073/pnas.1417566112

Figure Lengend Snippet: Keap1 and BTrCP levels are significantly linked to rodent MLSP and likely contribute to longevity-associated up-regulation in Nrf2-signaling activity. (A and B) Both Keap1 (A; P = 0.0013) and βTrCP (B; P = 0.0037) were negatively correlated with MLSP in rodents (n = 4 for each of 10 species). Both of these proteins negatively regulate Nrf2 by binding to Nrf2 and targeting it for degradation. (C) Using immunoprecipitation, we confirmed that Keap1 and Nrf2 were bound to Nrf2 in liver tissues of both mice and naked mole-rats (n = 6). Error bars represent SEM.

Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

Techniques: Activity Assay, Binding Assay, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

doi: 10.1073/pnas.1417566112

Figure Lengend Snippet: Nrf2 signaling is regulated by several distinct mechanisms. Nrf2 activity is augmented by exogenous (i.e., xenobiotic compounds) and/or endogenous (i.e., ROS) stressors. Free Nrf2 can bind to the ARE in the nucleus and activates hundreds of cytoprotective molecules, including detoxification enzymes like GST, antioxidant enzymes (e.g., NQO1), proteasome subunits, and molecular chaperones. Nrf2 activity is largely negatively regulated by Keap1, which binds to Nrf2 and targets it for ubiquitination and proteasomal degradation. Recently, other proteins have been shown to influence Nrf2 activity either by interacting directly with Nrf2 or with Keap1. Positive regulators, including p21 and p38MAPK, directly activate Nrf2 signaling, whereas P62/SQSTM1, PALB2, WTX, and DPP3 interact with Keap1 and thereby activate Nrf2 signaling. Other negative regulators of Nrf2, including βTrCP, SIAH2, and CRIF1, directly target it for degradation independent of Keap1. CRIF1 regulates Nrf2 signaling independent of the cell’s redox status. All of these molecules may be potential targets for cancer prevention or longevity therapeutics.

Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

Techniques: Activity Assay

Journal: International journal of molecular sciences

Article Title: Krüppel-like Factor 15 Suppresses Ferroptosis by Activating an NRF2/GPX4 Signal to Protect against Folic Acid-Induced Acute Kidney Injury.

doi: 10.3390/ijms241914530

Figure Lengend Snippet: Figure 6. KLF15 post-transcriptionally activates NRF2. (A,B) NRF2 expression was analyzed in KLF15-overexpressing or KLF15 knockdown HK2 cells by Western blotting. (C). NRF2 and GPX4

Article Snippet: The filters were then incubated with NRF2 antibody and protein A-agar beads (Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C for 1 h to produce an immunoprecipitated (IP) sample.

Techniques: Expressing, Knockdown, Western Blot

Journal: International journal of molecular sciences

Article Title: Krüppel-like Factor 15 Suppresses Ferroptosis by Activating an NRF2/GPX4 Signal to Protect against Folic Acid-Induced Acute Kidney Injury.

doi: 10.3390/ijms241914530

Figure Lengend Snippet: Figure 7. KLF15 attenuates the ubiquitin-mediated degradation of NRF2. (A–D) KLF15 knockdown HK2 and HEK 293 cells and control cells were treated with 10 µM of MG132 or DMSO for 2 h, and Western blot analysis was then performed to assess the protein expression of KLF15, NRF2, and GAPDH (loading control). KLF15, Krüppel-like factor 15; NRF2, nuclear factor erythroid 2-related factor 2; HK2, human tubular epithelial cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The filters were then incubated with NRF2 antibody and protein A-agar beads (Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C for 1 h to produce an immunoprecipitated (IP) sample.

Techniques: Ubiquitin Proteomics, Knockdown, Control, Western Blot, Expressing

Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for immunoblot analysis: anti-DDI2 (from our lab, produced from human DDI2 immunized-rabbit serum using HiTrap NHS activated HP columns, GE Healthcare, Chicago, IL, USA), anti-NRF1 (8052) and anti-NRF2 (12,721) from Cell Signaling (Danvers, MA, USA), anti-Tubulin (AG-27B-0005-C100) from AdipoGen (Epalinges, Switzerland), anti-MT (ADI-SPA-550-D) from Enzo Life Sciences (Farmingdale, NY, UA).

Techniques: Recombinant, Purification, Reverse Transcription, Bicinchoninic Acid Protein Assay, Activity Assay, MTS Assay, Gene Expression, Western Blot, Plasmid Preparation, Software

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (IP) of SIRT2 and NRF2 in extracts of hearts from wild-type (WT) mice. ( B ) Endogenous NRF2 acetylation levels in the hearts of WT and Sirt2 -/- mice at the baseline. Acetylated proteins were IPed by anti-acetyl antibody followed by immunoblotting with anti-NRF2 antibody. ( C ) NRF2 protein levels in neonatal rat cardiomyocytes (NRCMs) treated with Sirt2 siRNA. ( D ) NRF2 protein levels in H9c2 cells treated with control or Sirt2 siRNA and harvested at different time points after treatment with 100 µg/ml of CHX. ( E ) NRF2 protein levels in the nucleus in NRCMs treated with control or Sirt2 siRNA. ( F–H ) mRNA levels of NRF2 target genes in pentose phosphate pathway ( F ), quinone and glutathione-based detoxification ( G ), thioredoxin production ( H ) in H9c2 cells overexpressing empty vector (white bars) or SIRT2 (gray bars). *p<0.05 by Student’s t-test. Figure 5—source data 1. mRNA with overexpression of EV or SIRT2 as shown in . Figure 5—source data 2. mRNA with overexpression of EV or SIRT2 as shown in . Figure 5—source data 3. mRNA with overexpression of EV or SIRT2 as shown in . Figure 5—source data 4. Uncropped gels for . Figure 5—source data 5. Uncropped gels for unedited. Figure 5—source data 6. Uncropped gels for unedited.

Article Snippet: Antibody , Rabbit mAb NRF2 antibody , Cell Signaling Technology , Rabbit mAb #20733 , WB (1:1000).

Techniques: Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Over Expression

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet: ( A ) NRF2 protein levels in HL-1 cells treated with Sirt2 siRNA. ( B–D ) mRNA levels of NRF2 target genes in pentose phosphate pathway ( B ), quinone and glutathione-based detoxification ( C ), thioredoxin production ( D ) in HL-1 cells overexpressing empty vector (white bars) or SIRT2 (gray bars). *p<0.05 by Student’s t-test. Figure 5—figure supplement 2—source data 1. Uncropped gels for . Figure 5—figure supplement 2—source data 2. Uncropped gels for unedited. Figure 5—figure supplement 2—source data 3. mRNA with overexpression of EV or SIRT2 as shown in .

Article Snippet: Antibody , Rabbit mAb NRF2 antibody , Cell Signaling Technology , Rabbit mAb #20733 , WB (1:1000).

Techniques: Plasmid Preparation, Over Expression

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet: Ejection fraction (EF) ( A ) and fractional shortening (FS) ( B ) in wild-type (WT), Sirt2 -/- , and Sirt2 -/- /Nrf2 -/- double knockout (KO) mice 28 days after ischemia-reperfusion (I/R) (N=4–5). ( C ) Protocol for treatment of mice with SIRT2 inhibitor, AGK2. ( D ) Echo images of hearts from WT mice treated with either vehicle or AGK2. ( E–J ) EF ( E ), FS ( F ), left ventricular diameter during diastole (LVDd) ( G ), left ventricular diameter during systole (LVDs) ( H ), IVSd ( I ), and posterior wall thickness during diastole (PWTd) ( J ) in WT mice treated with AGK after trans-aortic constriction (TAC) according to the protocol in panel C (N=6–10). *p<0.05 by ANOVA for panels A–B or Student’s t-test for panels E–J. Figure 6—source data 1. Ejection fraction (EF) in wild-type (WT), Sirt2 -/- , and Sirt2 -/- / Nrf2 -/- mice after ischemia-reperfusion (I/R) as shown in . Figure 6—source data 2. Fractional shortening (FS) in wild-type (WT), Sirt2 -/- , and Sirt2 -/- / Nrf2 -/- mice after ischemia-reperfusion (I/R) as shown in . Figure 6—source data 3. Ejection fraction (EF) with AGK2 as shown in . Figure 6—source data 4. Fractional shortening (FS) with AGK2 as shown in . Figure 6—source data 5. Left ventricular diameter during diastole (LVDd) with AGK2 as shown in . Figure 6—source data 6. LVDs with AGK2 as shown in . Figure 6—source data 7. IVSd with AGK2 as shown in . Figure 6—source data 8. Posterior wall thickness during diastole (PWTd) with AGK2 as shown in .

Article Snippet: Antibody , Rabbit mAb NRF2 antibody , Cell Signaling Technology , Rabbit mAb #20733 , WB (1:1000).

Techniques: Double Knockout

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit mAb NRF2 antibody , Cell Signaling Technology , Rabbit mAb #20733 , WB (1:1000).

Techniques: Knock-Out, Activity Assay, In Vivo, Protease Inhibitor, Transfection, SYBR Green Assay, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Extraction, Staining, Sequencing, Recombinant, Plasmid Preparation, Software