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

Article Title: Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

doi: 10.1073/pnas.2217128120

Figure Lengend Snippet: Fig. 1. SSH1 inhibits Nrf2/ARE target gene expression independent of SSH1 phosphatase activity. (A1) Schematic of the Nrf2 reporter construct pREP-8xARE- GFP-SV40-BFP. (A2) Schematic of SSH1 and SSH1-CS proteins showing the catalytic domain (CAT) and binding sites for cofilin and p62. (B) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue), with vector control, myc-Nrf2, and/or Flag-SSH1 (red). (C) Quantification of Nrf2 reporter [one- way ANOVA, F (2, 218) = 18.62, P < 0.0001, post hoc Dunnett, ****P < 0.0001, **P = 0.0076, n = 15 to 20 images/condition/experiment from three experiments]. (D) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue) and vector or Flag-SSH1 (red), ± 200 μM H2O2 (2 h). (E) Quantification of Nrf2 reporter [one-way ANOVA, F (2, 519) = 124.7, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 15 to 20 images/condition/experiment from four experiments]. (F) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue), vector, Flag-SSH1, or Flag-SSH1CS (red), ± 250 μM H2O2 (3 h). (G) Quantification of Nrf2 reporter [one-way ANOVA, F (3, 308) = 61.03, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ns = not significant, n = 8 to 12 images/ condition/experiment from four experiments]. (H) Representative images of HT22 cells cotransfected with the Nrf2 reporter (green and blue) and control siRNA or SSH1 siRNA, stained for SSH1 (red), ± 250 μM H2O2 (3 h). (I) Quantification of Nrf2 reporter [one-way ANOVA, F (3, 855) = 41.31, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 15 to 20 images/condition/experiment from four experiments]. (J) Representative immunoblots from lysates of HEK293T cells expressing vector or Flag-SSH1 ± 15 µM NaAsO2 (18 h). (K) Quantification of HMOX1 and NQO1 proteins [one-way ANOVA; HMOX1: F (2, 21) = 310.2, P < 0.0001; NQO1: F (2, 21) = 34.82, P < 0.0001; post hoc Dunnett, ****P < 0.0001, ***P < 0.001, *P < 0.05. n = 8 samples/condition]. (L) Representative immunoblots from lysates of HEK293T cells coexpressing vector or myc-Nrf2 plus vector, Flag-SSH1, or Flag-SSH1-CS. (M) Quantification of HMOX1 protein [one-way ANOVA, F (2, 6) = 32.61, P = 0.0006, post hoc Dunnett, ***P = 0.006, **P = 0.0022, ns = not significant, n = 3 samples/condition]. (N) Representative immunoblots from lysates of HEK293T cells transfected with control or SSH1 siRNA ± 15 µM NaAsO2 (18 h). (O) Quantification of HMOX1 protein [one-way ANOVA, F (2, 15) = 185.4, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 6 samples/condition]. (P) Quantification of NQO1 protein [one-way ANOVA, F (2, 14) = 34.49, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ns = not significant, n = 6 samples/condition].

Article Snippet: The following antibodies were used to probe target proteins: rabbit polyclonal anti- SSH1(ECM Biosciences, SP1711); mouse monoclonal anti- Nrf2 (3G7)(Novus Biologicals, AF4000); rabbit monoclonal anti- Keap1 (D6B12) (Cell Signaling Technologies, 8,047); rabbit monoclonal anti- SQSTM1/p62 (D10E10) (Cell Signaling Technologies, 7,695); mouse monoclonal anti- FLAG (M2) (Sigma- Aldrich, F3165); rabbit monoclonal anti- Nrf2 (D1Z9C) (Cell Signaling Technologies, 12,721); mouse monoclonal anti- Keap1 (1B4) (Abcam, ab119403); mouse monoclonal antiSSH1 (1A5C8) (Santa Cruz Biotech, sc- 517226); Alexa Fluor 594 goat anti- rabbit IgG (Invitrogen, A11037); Alexa Fluor 594 goat anti- mouse IgG (Invitrogen, A11032); and Alexa Fluor 488 goat anti- rabbit IgG (Invitrogen, A11034).

Techniques: Targeted Gene Expression, Activity Assay, Construct, Binding Assay, Plasmid Preparation, Control, Staining, Western Blot, Expressing, Transfection

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

Article Title: Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

doi: 10.1073/pnas.2217128120

Figure Lengend Snippet: Fig. 3. AD and FTLD-tau brains exhibit excessive levels of inhibitory SSH1–Nrf2 and Keap1–Nrf2 interactions. (A) Representative images of HT22 cells transfected with GFP (green) and vector or Flag-SSH1, treated ± 8 µM NaAsO2 (14 h) and subjected to PLA for SSH1–Nrf2 (red). (B) Quantification of SSH1–Nrf2 PLA puncta area/cell [Brown-Forsythe and Welch ANOVA, F (3, 202.7) = 30.48, F (3, 183.2) = 62.13, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 10 to 15 images/ condition/experiment from four experiments]. (C) Representative images of human frontal gyrus sections from nondementia, AD, and FTLD-tau cases showing DAPI (blue) and PLA for SSH1–Nrf2 (red) and Keap1–Nrf2 (red). (D and E) Quantification of SSH1–Nrf2 PLA area in (D) control vs. AD (two-tailed t test, t = 6.329, df = 156, ****P < 0.0001, n = 8 to 10 images/case from 7 to 8 case/condition) and (E) control vs. FTLD-tau (two-tailed t test, t = 2.353, df = 157, *P = 0.0199, n = 8 to 10 images/case from eight cases/condition). (F and G) Quantification of Keap1–Nrf2 PLA area in (F) control vs. AD (two-tailed t test, t = 3.371, df = 154, ***P = 0.0009; n = 8 to 10 images/case from 7 to 8 cases/condition) and (G) control vs. FTLD-tau (two-tailed t test, t = 4.752, df = 152; ****P < 0.0001; n = 8 to 10 images/ case from eight cases/condition).

Article Snippet: The following antibodies were used to probe target proteins: rabbit polyclonal anti- SSH1(ECM Biosciences, SP1711); mouse monoclonal anti- Nrf2 (3G7)(Novus Biologicals, AF4000); rabbit monoclonal anti- Keap1 (D6B12) (Cell Signaling Technologies, 8,047); rabbit monoclonal anti- SQSTM1/p62 (D10E10) (Cell Signaling Technologies, 7,695); mouse monoclonal anti- FLAG (M2) (Sigma- Aldrich, F3165); rabbit monoclonal anti- Nrf2 (D1Z9C) (Cell Signaling Technologies, 12,721); mouse monoclonal anti- Keap1 (1B4) (Abcam, ab119403); mouse monoclonal antiSSH1 (1A5C8) (Santa Cruz Biotech, sc- 517226); Alexa Fluor 594 goat anti- rabbit IgG (Invitrogen, A11037); Alexa Fluor 594 goat anti- mouse IgG (Invitrogen, A11032); and Alexa Fluor 488 goat anti- rabbit IgG (Invitrogen, A11034).

Techniques: Transfection, Plasmid Preparation, Control, Two Tailed Test

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

Article Title: Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

doi: 10.1073/pnas.2217128120

Figure Lengend Snippet: Fig. 4. Ssh1 elimination increases nuclear Nrf2, reduces oxidative injury, and alleviates AD pathology. (A) Representative images of the cortex from 7-mo-old WT, P301S, and P301S;Ssh1−/− mice stained for Nrf2 (green) and DAPI (blue). White boxes serially magnified to the right. (B) Quantification of nuclear/cytoplasmic Nrf2 intensity [one-way ANOVA, F (2, 134) = 13.06, P < 0.0001, post hoc Dunnett, ****P < 0.0001, **P = 0.0013, n = 10 to 12 images/mouse from four mice/genotype]. (C) Representative images of the cortex and hippocampus (CA3) stained for 8-OHdG (green) and DAPI (blue) from 7-mo-old WT, P301S, and P301S;Ssh1−/− mice. (D and E) Quantification of 8-OHdG intensity in the (D) cortex [one-way ANOVA, F (2, 134) = 37.79, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 10 to 13 images/mouse from four mice/genotype) and (E) hippocampus [one-way ANOVA, F (2, 70) = 17.85, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ***P = 0.0003, n = 5 to 7 images/mouse from four mice/genotype]. (F) Representative images of the cortex from 8-mo-old WT, APP/PS1, and APP/PS1;Ssh1−/− mice stained for 8-OHdG (red) and DAPI (blue). (G) Quantification of 8-OHdG intensity [one-way ANOVA, F (2, 181) = 36.83, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 10 to 12 images/mouse from four mice/genotype]. (H) Representative images of silver staining of the cortex and hippocampus from 7-mo-old WT, P301S, and P301S;Ssh1−/− mice. Red arrows indicate silver-positive degenerating axons. (I and J) Quantification of silver-positive axons in the (I) cortex [one-way ANOVA, F (2, 21) = 14.5, P = 0.0001, post hoc Dunnett, ***P < 0.0005, n = 6 to 10 mice/genotype] and (J) hippocampus [one-way ANOVA, F (2, 21) = 7.545, P = 0.0034, post hoc Dunnett, **P < 0.0081, n = 6 to 10 mice/genotype]. (K) Representative images of the cortex and hippocampus (CA3) stained for pS199/202-tau (green) and DAPI (blue) from 7-mo-old P301S and P301S;Ssh1−/− mice. (L and M) Quantification of pS199/202-tau intensity in the (L) cortex (two-tailed t test, t = 13.85, df = 221, ****P < 0.0001, n = 20 to 30 images/mouse from four mice/genotype) and (M) hippocampus (two-tailed t–test, t = 8.147, df = 104, ****P < 0.0001, n = 13 to 18 images/mouse from four mice/genotype). (N) Representative images of the cortex and hippocampus stained for Aβ (green) and DAPI (blue) from 8-mo-old WT, APP/PS1, and APP/PS1;Ssh1−/− mice. (O) Quantification of Aβ intensity in the cortex (two-tailed t test, t = 2.784, df = 76, **P = 0.0068, n = 6 to 8 images/mouse from 4 to 6 mice/genotype).

Article Snippet: The following antibodies were used to probe target proteins: rabbit polyclonal anti- SSH1(ECM Biosciences, SP1711); mouse monoclonal anti- Nrf2 (3G7)(Novus Biologicals, AF4000); rabbit monoclonal anti- Keap1 (D6B12) (Cell Signaling Technologies, 8,047); rabbit monoclonal anti- SQSTM1/p62 (D10E10) (Cell Signaling Technologies, 7,695); mouse monoclonal anti- FLAG (M2) (Sigma- Aldrich, F3165); rabbit monoclonal anti- Nrf2 (D1Z9C) (Cell Signaling Technologies, 12,721); mouse monoclonal anti- Keap1 (1B4) (Abcam, ab119403); mouse monoclonal antiSSH1 (1A5C8) (Santa Cruz Biotech, sc- 517226); Alexa Fluor 594 goat anti- rabbit IgG (Invitrogen, A11037); Alexa Fluor 594 goat anti- mouse IgG (Invitrogen, A11032); and Alexa Fluor 488 goat anti- rabbit IgG (Invitrogen, A11034).

Techniques: Staining, Silver Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62

doi: 10.1038/s41598-019-50877-8

Figure Lengend Snippet: TFEB increases the transcriptional activity and the nuclear localization of Nrf2. ( a ) The gene expression levels in H4 cells (Control) and H4 cells stably expressing TFEB (TFEB) were analyzed using cDNA microarray as described in Methods. Heat map shows the expression levels for Nrf2-response genes. Shades of red represent up-regulation, and shades of green represent down-regulation. ( b ) H4 cells (Control) and H4 cells stably expressing TFEB (TFEB) were transiently transfected with the ARE-Luc reporter plasmid and assayed for the luciferase activity. ( c ) H4 cells stably expressing TFEB fixed with 4% paraformaldehyde were immunostained using anti-TFEB and Nrf2 antibodies. Fluorescence signals were observed using a fluorescence microscope. ( d ) Nuclear (NF) and cytosolic (CF) fractions from control and TFEB cells were prepared as described in Methods. The cellular locations of TFEB and Nrf2 were analyzed by immunoblotting using anti-TFEB and Nrf2 antibodies, respectively. To examine the purity of the fractionations, the blot was probed with antibodies to lamin (A/C), a marker for nuclear fraction, and tubulin, a marker for cytosolic fraction. Full blots are provided in Supplementary Fig. . Data shown are mean ± S.E. of four independent experiments and were analyzed using Student’s t test. (** p < 0.01).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activity Assay, Gene Expression, Control, Stable Transfection, Expressing, Microarray, Transfection, Plasmid Preparation, Luciferase, Fluorescence, Microscopy, Western Blot, Marker

Journal: Scientific Reports

Article Title: TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62

doi: 10.1038/s41598-019-50877-8

Figure Lengend Snippet: TFEB increases the expression of Nrf2-response genes. ( a ) The protein levels of Nrf2-response genes in H4 cells (Control) and H4 cells stably expressing TFEB (TFEB) were analyzed by immunoblotting using anti-TFEB, Nrf2, HO-1, GSTM1, p62, and NDP52 antibodies, respectively. Full blots are provided in Supplementary Fig. . ( b ) The mRNA levels of Nrf2-response genes were analyzed by qRT-PCR as described in Methods. Bar graph represents the relative mRNA level of genes in TFEB cells compared to H4 cells. Data shown are mean ± S.E. of three independent experiments and were analyzed using Student’s t test. (* p < 0.05; *** p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Expressing, Control, Stable Transfection, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62

doi: 10.1038/s41598-019-50877-8

Figure Lengend Snippet: TFEB increases the stability of Nrf2. ( a ) The protein levels of TFEB and Nrf2 in H4 cells (Control) and H4 cell lines stably expressing TFEB (TFEB) were analyzed by immunoblotting using each corresponding antibody, respectively. ( b ) Dot plot represents the relative ratio of Nrf2 normalized with that of actin. ( c ) To examine the ubiquitination level of Nrf2 in H4 and TFEB cells, the cells were treated with 5 µM of MG132, a proteasome inhibitor, for 24 h. The cell lysates were used for the immunoprecipitation of Nrf2 using anti-Nrf2 antibody. The ubiquitination levels of Nrf2 were examined by immunoblotting using anti-ubiquitin antibody. ( d ) The cells were kept with or without 100 µM of cycloheximide (CHX) for 30 min. The protein levels of Nrf2 were analyzed by immunoblotting using anti-Nrf2 antibody. ( e ) Dot plot represents the relative ratio of Nrf2 in cells treated with CHX in contrast to those not treated. ( f ) HEK293 cells were transiently transfected with the Myc-Nrf2 expression plasmid and together with or without the pHM6-TFEB expression plasmid. The protein levels of Nrf2 and TFEB were analyzed by immunoblotting using anti-Myc and TFEB antibodies, respectively. The asterisk on the Nrf2 panel indicates a non-specific band. Full blots are provided in Supplementary Fig. . Data shown are mean ± S.E. of four independent experiments and were analyzed using Student’s t test. (* p < 0.05; *** p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Control, Stable Transfection, Expressing, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62

doi: 10.1038/s41598-019-50877-8

Figure Lengend Snippet: TFEB represses the expression of DCAF11. ( a ) The mRNA levels of Keap1, β-TrCP, DCAF11 and Hrd1 were analyzed by qRT-PCR as described in Methods. Bar graph represents the relative mRNA levels of Keap1 and Nrf2-specific E3 ubiquitin ligase genes in H4 cells stably expressing TFEB (TFEB) compared to H4 cells (Control). ( b ) The protein levels of Keap1 and Nrf2-specific E3 ubiquitin ligases in H4 and TFEB cells were analyzed by immunoblotting using each corresponding antibody. Full blots are provided in Supplementary Fig. . Data shown are mean ± S.E. of three independent experiments and were analyzed using Student’s t test. (** p < 0.01; *** p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Expressing, Quantitative RT-PCR, Ubiquitin Proteomics, Stable Transfection, Control, Western Blot

Journal: Scientific Reports

Article Title: TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62

doi: 10.1038/s41598-019-50877-8

Figure Lengend Snippet: The phosphorylation of p62 at S349 is involved in TFEB-mediated Nrf2 activation. ( a ) The levels of phosphorylated p62 at S349 and total p62 in H4 cells (Control) and H4 cells stably expressing TFEB (TFEB) were analyzed by immunoblotting using anti-phospho-specific p62 (S349) and anti-p62 antibodies, respectively. ( b ) HEK 293 cells with the wild type or haplotype (+/−) p62 gene were transiently transfected with the Myc-Nrf2 expression plasmid and together with or without the pHM6-TFEB expression plasmid. The protein levels of Nrf2, phosphorylated p62 at S349, p62 and TFEB were analyzed by immunoblotting using anti-Myc, phospho p62 (S349), p62 and HA antibodies. The asterisk on the Nrf2 panel indicates a non-specific band. ( c ) Scatter plot represents the relative ratio of Nrf2 and phosphorylated p62 at S349 normalized with that of actin. ( d ) H4 cells stably expressing TFEB were transiently transfected with the ARE-Luc reporter plasmid along with the wild-type or mutant p62 (S349A) plasmid. The number under graph indicates the amount of plasmids transfected in each well. The luciferase activity in the cells was measured following 24 h incubation. Full blots are provided in Supplementary Fig. . Data shown are mean ± S.E. of four independent experiments and were analyzed using Student’s t test. (*** p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Activation Assay, Control, Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62

doi: 10.1038/s41598-019-50877-8

Figure Lengend Snippet: Schematic diagram showing the mechanism of Nrf2 activation by TFEB. TFEB first stabilizes Nrf2 through the repression of DCAF11, its E3 ubiquitin ligase, and activates Nrf2, resulting in the increased expression and phosphorylation of p62 at S349. Then, increased phosphorylated p62 disrupts the binding of Keap1 and Nrf2, thus leading to sustained Nrf2 activation through a positive feedback loop.

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Ubiquitin Proteomics, Expressing, Phospho-proteomics, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Nrf2 activation restores, while Nrf2 suppression mimics, hyperglycemia-induced ROS generation. (A) Rat macrophages were infected by an empty vector (EMP), Nrf2 lentivirus (Nrf2↑), or Nrf2 knockdown lentivirus (shNrf2) in the presence of either 5 µM Nrf2 activator DMF or 5 µM Nrf2 inhibitor ML385 (with 0.1% DMSO as vehicle) and then incubated in either low glucose (5 mM) or high glucose (25 mM glucose) continuously for 5 days in the presence of 1% FBS, and the cells were harvested on each day for analysis. (A) ROS generation in the presence of Nrf2 overexpression (Nrf2↑) or Nrf2 activator (DMF), n = 5. * P < 0.05, vs day 0 group; †, P < 0.05, vs day 3 group. (B) ROS generation in the presence of Nrf2 knockdown (shNrf2) or Nrf2 inhibitor (ML385), n = 5. * P < 0.05, vs day 0 group; † P < 0.05, vs day 3 group. (C) 3-Nitrotyrosine formation on day 5 treatment, n = 5. * P < 0.05, vs LG/EMP group. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Infection, Plasmid Preparation, Knockdown, Incubation, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Nrf2 activation increases, while Nrf2 suppression decreases, the expression of NQO1 and HO1. (A) Rat macrophages were incubated in hyperglycemia (25 mM glucose) continuously for 5 days in the presence of 1% FBS, and the cells were used for gene analysis, n = 4. * P < 0.05, vs day 0 group. (B – H) Rat macrophage cells were infected by an empty vector (EMP), Nrf2 lentivirus (Nrf2↑), or Nrf2 knockdown lentivirus (shNrf2) in the presence of either 5 µM Nrf2 activator DMF or 5 µM Nrf2 inhibitor ML385 (with 0.1% DMSO as vehicle), and then incubated in either low glucose (5 mM) or high glucose (25 mM glucose) continuously for 5 days in the presence of 1% FBS. The cells were harvested on day 5 for analysis. * P < 0.05, vs LG/EMP group. (B) mRNA analysis by qPCR, n = 4. (C) Representative picture for Western blots. (D) Protein quantitation for (C) , n = 5. (E) Representative pictures for IP/WB by Maf/Nrf2. (F) Protein quantitation for (E) , n = 5. (G) ChIP by Nrf2 antibody on NQO1 and HO1 promoter, n = 4. (H) Reporter activity assay for NQO1 and HO1, n = 5. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Expressing, Incubation, Infection, Plasmid Preparation, Knockdown, Western Blot, Protein Quantitation, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Activation of Nrf2 prevents diabetes-induced oxidative stress and expression of proinflammatory cytokines in rats. The burn injuries were introduced, and the experimental rats were divided into four groups: control rats (CTL/VEH), STZ diabetic rats (STZ/VEH), STZ rats treated by 15 mg/kg/d of DMF (STZ/DMF), or CTL rats received 15 mg/kg/d of ML385 (CTL/ML385). The wounds were excised and collected from the experimental rats for further analysis. (A) Superoxide anion release, n = 5. (B) mRNA levels for Nrf2 and its target genes by qPCR, n = 4. (C) Representative pictures for Western blots. (D) Protein quantitation for (C) , n = 4. (E) Representative pictures for NQO1 expression by immunohistochemistry. (F) Representative pictures for HO1 expression by immunohistochemistry. (G) mRNA levels for proinflammatory cytokines, n = 4. (H) IL1β in serum, n = 5. (I) IL6 in serum, n = 5. (J) MCP1 in serum, n = 5. * P < 0.05, vs CTL group; ¶ P < 0.05, vs STZ/VEH group. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Expressing, Control, Western Blot, Protein Quantitation, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Expression of Nrf2/HO1 prevents, while knockdown of Nrf2/HO1 mimics, hyperglycemia-induced expression of proinflammatory cytokines. Rat macrophages were infected by an empty vector (EMP), Nrf2 lentivirus (Nrf2↑), HO1 lentivirus (HO1↑), Nrf2 knockdown (shNrf2), or HO1 knockdown lentivirus (shHO1) and then incubated in either control (CTL) or 5 ng/mL LPS, and the cells were harvested on day 5 for analysis. (A) mRNA levels by qPCR, n = 4. * P < 0.05, vs LPS/EMP group; ¶ P < 0.05, vs LPS/Nrf2↑ group. (B) IL1β secretion, n = 5. * P < 0.05, vs CTL/EMP group; ¶ P < 0.05, vs LPS/EMP group. (C) IL6 secretion, n = 5. * P < 0.05, vs CTL/EMP group; ¶ P < 0.05, vs LPS/EMP group. (D) MCP1 secretion, n = 5. * P < 0.05, vs CTL/EMP group. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Knockdown, Infection, Plasmid Preparation, Incubation, Control

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Nrf2 activator DMF prevents, while Nrf2 blocker ML385 mimics, hyperglycemia-induced expression of proinflammatory cytokines. Rat macrophage cells were treated by either 5 µM Nrf2 activator DMF or 5 µM Nrf2 inhibitor ML385, then incubated in either low glucose (5 mM) or high glucose (25 mM glucose) continuously for 5 days, and the cells were harvested on day 5 for further analysis. (A) mRNA levels by qPCR, n = 4. (B) IL1β secretion, n = 5. (C) IL6 secretion, n = 5. (D) MCP1 secretion, n = 5. * P < 0.05, vs LG/VEH group; # P < 0.05, vs HG/VEH group. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Incubation

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Nrf2 activation accelerates wound healing in diabetic rats, while Nrf2 inhibitor mimics the effect of diabetes. The experimental rats were divided into four groups: control rats (CTL/VEH), STZ diabetic rats (STZ/VEH), STZ rats treated by 15 mg/kg/d of DMF (STZ/DMF), or CTL rats received 15 mg/kg/d of ML385 (CTL/ML385), for further analysis. (A) Photographs of representative wounds on day 12 after burns. (B) Quantitation of burn area on day 18, n = 8. (C) Graphical depiction of wound areas on different days after burns, n = 8. (D) Representative pictures for IP/WB by Maf/Nrf2. (E) Protein quantitation for (D) , n = 5. (F) NAG activity as the marker of macrophage accumulation, n = 5. (G) Hematoxylin-eosin stains of wound tissues on day 12 after burns with occurrence of granulation tissue in the wounds. (H) Deposition of granulation tissues, n = 8. (I) The representative pictures for evaluation of vascularity (assessed by CD31 immunohistochemistry) on day 12 after burns. (J) Numbers of CD31 positive vessels per high-power field area on day 12 after burns, n = 8. * P < 0.05, vs CTL/VEH group; ¶ P < 0.05, vs STZ/VEH group. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Control, Quantitation Assay, Protein Quantitation, Activity Assay, Marker, Immunohistochemistry

Journal: Redox Biology

Article Title: Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

doi: 10.1016/j.redox.2024.103117

Figure Lengend Snippet: List of primary antibodies used in the study.

Article Snippet: NRF2 , Cell Signalling Technologies , 20733 , Lot 1 , Rabbit , 1:1000.

Techniques:

Journal: Redox Biology

Article Title: Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

doi: 10.1016/j.redox.2024.103117

Figure Lengend Snippet: List of probe based qPCR assays used in the study.

Article Snippet: NRF2 , Cell Signalling Technologies , 20733 , Lot 1 , Rabbit , 1:1000.

Techniques: Amplification

Journal: Redox Biology

Article Title: Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

doi: 10.1016/j.redox.2024.103117

Figure Lengend Snippet: a, b) INS-1 832/13 cells were treated for 24 h with DMSO, the GSK3 inhibitors CT99021 (5 μmol/l) or LY2090314 (20 nmol/l), the KEAP1 inhibitors TBE31 (50 nmol/l) or SFN (5 μmol/l), or combinations (CT99021+TBE31, LY2090314+TBE31 or CT99021+SFN). Whole-cell protein lysates from three independent experiments (20 μg protein/lane) were analysed by western blotting using β-tubulin as a loading control. c) INS-1 832/13 cells were treated for 24 h with DMSO, CT99021 (5 μmol/l), TBE31 (50 nmol/l) or CT99021 +TBE31, and then cytoplasmic and nuclear protein extracts were prepared. Samples of either cytoplasmic fractions (20 μg protein/lane) or nuclear fractions (10 μg protein/lane) were separated by SDS-PAGE and NRF2 protein levels analysed with TBP as loading control. Data from seven independent experiments were analysed by paired ANOVA with Sidak's post hoc test. d,e) RNA from INS-1 832/13 cells (n = 3) or islets isolated from 12-week-old male C57BL/6J mice (n = 6) were extracted after 24 h treatment with the agents described above and expression of NRF2-target genes analysed by qPCR. Data are presented as mean and standard error and analysed by one-way ANOVA with Sidak's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: NRF2 , Cell Signalling Technologies , 20733 , Lot 1 , Rabbit , 1:1000.

Techniques: Western Blot, Control, SDS Page, Isolation, Expressing

Journal: Redox Biology

Article Title: Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

doi: 10.1016/j.redox.2024.103117

Figure Lengend Snippet: a) INS-1 832/13 stable cell lines lacking KEAP1 were generated by transfection with a lentiviral all-in-one plasmid containing either control/Scramble gRNA or rat KEAP1 gRNA along with Cas9, and a puromycin selection gene. Whole-cell lysates were extracted and KEAP1 deletion as well as subsequent enhanced NRF2 expression and activity (NQO1) were confirmed by western blotting. Tubulin was used as a loading control. b) GFP or KEAP1 KO cells were transfected with siRNA targeting GSK3α, GSK3β or both GSK3α+β. Seventy-two hr after transfection, whole cell lysates were extracted and analysed by western blot as shown. This confirmed GSK3α+β knockdown and the subsequent impact on phosphorylation of the GSK3 substrate CRMP2, along with NRF2 protein levels, assessed. Tubulin was used as loading control. The GSK3, pCRMP2 and NRF2 blots were quantified from 3 separate experiments run in duplicate and are presented relative to control as mean ± standard error, analysed by one-way ANOVA with Dunnett's post hoc test. c) KEAP1 KO or GFP expressing control cells were transfected with siRNA targeting GSK3α, GSK3β or both GSK3α+β. RNA was extracted 72h after transfection and NRF2-target gene expression was quantified by qPCR. Data are presented as mean ± standard error and analysed by one-way ANOVA with Dunnett's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: NRF2 , Cell Signalling Technologies , 20733 , Lot 1 , Rabbit , 1:1000.

Techniques: Stable Transfection, Generated, Transfection, Plasmid Preparation, Control, Selection, Expressing, Activity Assay, Western Blot, Knockdown, Phospho-proteomics, Targeted Gene Expression

Journal: Redox Biology

Article Title: Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

doi: 10.1016/j.redox.2024.103117

Figure Lengend Snippet: a) KEAP1 KO or GFP expressing control cells were transduced with adenoviral particles expressing GSK3α or GSK3β under the CMV promoter. Twenty-four hr later whole cell lysates were analysed for protein levels. This confirmed GSK3α+β overexpression and the subsequent impact on phosphorylation of the GSK3 substrate CRMP2, along with NRF2 protein levels, assessed. Tubulin was used as loading control. The GSK3, pCRMP2 and NRF2 blots were quantified from 3 separate experiments run in duplicate and are presented relative to control as mean ± standard error, analysed by one-way ANOVA with Dunnett's post hoc test. b) KEAP1 KO or GFP expressing control cells were transduced with GSK3α, GSK3β or GSK3α+β adenovirus, cultured for a further 24 h prior to RNA extraction and quantification of NRF2-target gene expression by qPCR. Data are presented as mean and standard error and analysed by one-way ANOVA with Dunnett's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: NRF2 , Cell Signalling Technologies , 20733 , Lot 1 , Rabbit , 1:1000.

Techniques: Expressing, Control, Transduction, Over Expression, Phospho-proteomics, Cell Culture, RNA Extraction, Targeted Gene Expression

Journal: Redox Biology

Article Title: Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

doi: 10.1016/j.redox.2024.103117

Figure Lengend Snippet: a) INS-1 832/13 cells were treated with DMSO, NRF2-βTrCP protein interaction inhibitor PHAR (10 μmol/l), KEAP1 inhibitor TBE31 (50 nmol/l) or PHAR + TBE31 for 24 h. Whole-cell protein lysates from three independent experiments (20 μg protein/lane) were analysed by western blotting using β-tubulin as a loading control. b) INS-1 832/13 cells were treated with the above agents for 24 h and cytoplasmic or nuclear fractions were extracted using NE-PER™ Kit. Nuclear fractions (10 μg protein) were separated by SDS-PAGE and NRF2 levels were analysed by western blotting using TBP as a loading control. Data from five independent experiments was analysed by paired ANOVA with Sidak's post hoc test. c,d) RNA was purified from INS-1 832/13 cells (n = 3), or islets from 12-week-old male C57BL/6J mice (n = 6), after 24 h treatment with the above agents and gene transcription analysed by qPCR. Data are presented as mean and standard error and analysed by one-way ANOVA with Sidak's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: NRF2 , Cell Signalling Technologies , 20733 , Lot 1 , Rabbit , 1:1000.

Techniques: Western Blot, Control, SDS Page, Purification

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of DJ-1 reduces oxidative stress and attenuates hypoxia/reoxygenation injury in NRK-52E cells exposed to high glucose

doi: 10.3892/ijmm.2016.2680

Figure Lengend Snippet: Effects of DJ-1 overexpression and NAC on the protein expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). (A, C and E) Western blot analysis results of DJ-1, Nrf2 and HO-1 protein expression in NRK-52E cells transfected with pEX-2-EGFP-DJ-1 and exposed to high glucose (HG) and hypoxia/reoxygenation (H/R); (B, D and F) western blot analysis results of DJ-1, Nrf2 and HO-1 protein in cells pre-treated with NAC and exposed to HG and H/R. β-actin was used for normalization of the total protein DJ-1 and HO-1 levels. Lamin B1 was used for normalization of the nuclear protein Nrf2. Data are expressed as the means ± SEM. * P<0.05, ** P<0.01, *** P<0.001 compared with the LG vector (empty vector) control group; # P<0.05, ## P<0.01, ### P<0.001 compared with HG vector control group; & P<0.05, && P<0.01 compared with HG + H/R vector control group. Results shown are representative of at least 3 independent experiments.

Article Snippet: The antibodies used are listed as follows: rabbit anti-rat DJ-1 monoclonal antibody (#5933; Cell Signaling Technology, Danvers, MA, USA); rabbit anti-rat Nrf2 (sc-722) and HO-1 (sc-10789) polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) rabbit anti-rat β-actin polyclonal antibody (GB13001; Wuhan Goodbio Technology Co., Ltd., Wuhan, China) and rabbit anti-rat Lamin B1 polyclonal antibody (BA1228; Boster, Wuhan, China).

Techniques: Over Expression, Expressing, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Cell Reports Medicine

Article Title: Tumor-immune microenvironment and NRF2 associate with clinical efficacy of PD-1 blockade combined with chemotherapy in lung squamous cell carcinoma

doi: 10.1016/j.xcrm.2023.101302

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for the immunoblotting analysis were: anti-NRF2 (1:1000; A11159; ABclonal), anti-PD-L1 (1:1000; E1L3N; 13684; Cell Signaling Technology), anti-NQO1 (1:1000; A0047; ABclonal), and anti-α-Tubulin (1:5000; sc-134237; Santa Cruz Biotechnology).

Techniques: Western Blot, Flow Cytometry, Recombinant, Transfection, Chromatin Immunoprecipitation, Immunohistochemistry, Gene Expression, Mutagenesis, Sequencing, Plasmid Preparation, Software

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Nrf2 activation increases, while Nrf2 suppression decreases, the expression of NQO1 and HO1. (A) Rat macrophages were incubated in hyperglycemia (25 mM glucose) continuously for 5 days in the presence of 1% FBS, and the cells were used for gene analysis, n = 4. * P < 0.05, vs day 0 group. (B – H) Rat macrophage cells were infected by an empty vector (EMP), Nrf2 lentivirus (Nrf2↑), or Nrf2 knockdown lentivirus (shNrf2) in the presence of either 5 µM Nrf2 activator DMF or 5 µM Nrf2 inhibitor ML385 (with 0.1% DMSO as vehicle), and then incubated in either low glucose (5 mM) or high glucose (25 mM glucose) continuously for 5 days in the presence of 1% FBS. The cells were harvested on day 5 for analysis. * P < 0.05, vs LG/EMP group. (B) mRNA analysis by qPCR, n = 4. (C) Representative picture for Western blots. (D) Protein quantitation for (C) , n = 5. (E) Representative pictures for IP/WB by Maf/Nrf2. (F) Protein quantitation for (E) , n = 5. (G) ChIP by Nrf2 antibody on NQO1 and HO1 promoter, n = 4. (H) Reporter activity assay for NQO1 and HO1, n = 5. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Expressing, Incubation, Infection, Plasmid Preparation, Knockdown, Western Blot, Protein Quantitation, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Activation of Nrf2 prevents diabetes-induced oxidative stress and expression of proinflammatory cytokines in rats. The burn injuries were introduced, and the experimental rats were divided into four groups: control rats (CTL/VEH), STZ diabetic rats (STZ/VEH), STZ rats treated by 15 mg/kg/d of DMF (STZ/DMF), or CTL rats received 15 mg/kg/d of ML385 (CTL/ML385). The wounds were excised and collected from the experimental rats for further analysis. (A) Superoxide anion release, n = 5. (B) mRNA levels for Nrf2 and its target genes by qPCR, n = 4. (C) Representative pictures for Western blots. (D) Protein quantitation for (C) , n = 4. (E) Representative pictures for NQO1 expression by immunohistochemistry. (F) Representative pictures for HO1 expression by immunohistochemistry. (G) mRNA levels for proinflammatory cytokines, n = 4. (H) IL1β in serum, n = 5. (I) IL6 in serum, n = 5. (J) MCP1 in serum, n = 5. * P < 0.05, vs CTL group; ¶ P < 0.05, vs STZ/VEH group. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Expressing, Control, Western Blot, Protein Quantitation, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation

doi: 10.3389/fphar.2019.01099

Figure Lengend Snippet: Expression of Nrf2/HO1 prevents, while knockdown of Nrf2/HO1 mimics, hyperglycemia-induced expression of proinflammatory cytokines. Rat macrophages were infected by an empty vector (EMP), Nrf2 lentivirus (Nrf2↑), HO1 lentivirus (HO1↑), Nrf2 knockdown (shNrf2), or HO1 knockdown lentivirus (shHO1) and then incubated in either control (CTL) or 5 ng/mL LPS, and the cells were harvested on day 5 for analysis. (A) mRNA levels by qPCR, n = 4. * P < 0.05, vs LPS/EMP group; ¶ P < 0.05, vs LPS/Nrf2↑ group. (B) IL1β secretion, n = 5. * P < 0.05, vs CTL/EMP group; ¶ P < 0.05, vs LPS/EMP group. (C) IL6 secretion, n = 5. * P < 0.05, vs CTL/EMP group; ¶ P < 0.05, vs LPS/EMP group. (D) MCP1 secretion, n = 5. * P < 0.05, vs CTL/EMP group. Data were expressed as mean ± SEM.

Article Snippet: The shRNA lentivirus plasmids for rat Nrf2 (sc-156128-SH), HO1 (sc-270124-SH), or nontarget control (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Knockdown, Infection, Plasmid Preparation, Incubation, Control

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: A , Titration of Keap1-mediated suppression of Nrf2 transcritional activity. NIH 3T3 cells were transfected with plasmids containing an ARE-dependent firefly luciferase reporter gene (ARE-Luc), expression plasmids for wild-type HA-Nrf2 and CBD-Keap1, and plasmids encoding Renilla luciferase to normalize transfection efficiency. The transfections were performed with 100 ng of ARE-Luc plasmid, 10 ng of pRL-TK, 40 ng of HA-Nrf2, and 1–40 ng of CBD-Keap1, as well as different amounts of pcDNA3.1 to maintain a total level of plasmid DNA at 200 ng. ARE reporter activity was measured 24 hours after the transfection. Results are representative of three independent experiments. * p <0.05 vs control pcDNA3.1. B , Effect of dh404 on Nrf2 nuclear translocation. NIH 3T3 cells were transfected for 24 hours with plasmids of HA-Nrf2 and CDB-Keap1 with a ratio of 10∶1 that resulted in complete suppression of Nrf2 transcriptional activity as indicated in A . The transfected cells were treated with dh404 (200 nM) in serum-free DMEM for 2 hours, and then subjected to immunofluorescence cofocal microscopic analysis. C , Cysteine residues in Keap1 required for dh404-mediated Nrf2 activation. NIH 3T3 cells were transfected for 24 hours with plasmids of HA-Nrf2 and CDB-Keap1 or plasmids of mutant Keap1 containing single cysteine-to-serine substitutions with a ratio of 10∶1 as indicated. The transfected cells were treated with or without dh404 (200 nM) in serum-free DMEM for 6 hours prior to ARE reporter assay. Results are representative of three independent experiments. * p <0.05 vs Nrf2 transfected group.

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Titration, Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Control, Translocation Assay, Immunofluorescence, Activation Assay, Mutagenesis, Reporter Assay

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: A , HEK 293 cells that were transfected with plasmids of HA-Nrf2 (1000 ng), CBD-Keap1 (100 ng) and HA-Ub (1000 ng) for 24 hours were treated with or without dh404 (200 nM) in DMEM with 10% FBS for 2 hours as indicated. Ubiquitination of Nrf2 was determined by immunoprecipitation (IP) and immunoblot (IB) analysis using appropriate antibodies as indicated. Inputs of 10 µg of whole cell lysates were utilized to monitor the protein expression levels of target genes. B & C , HEK 293 cells that were transfected with plasmids of HA-Nrf2 (800 ng) and CBD-Keap1 (80 ng) for 24 hours were treated with or without dh404 (200 nM) and/or CHX (10 µg/ml) in DMEM with 10% FBS as indicated. D, HEK293 Cells were transfected with HA-Nrf2, CBD-Keap1 or mutant Keap1 as described above, and then stimulated with or without dh404 (200 nM) for 2 hours. Whole cell lysates were subjected to immunoblot analysis using anti(α)Nrf2, αKeap1 or αGapdah, as indicated. Results are representative of three independent experiments.

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: HEK293 cells were transfected with plasmids of HA-Nrf2 (1000 ng), CBD-Keap1 (100 ng), HA-Cul3 (500 ng) or Myc-Rbx1 (400 ng) for 24 hours as indicated, and then were treated with or without dh404 (200 nM) in DMEM with 10% FBS for 2 hours as indicated. A , Recapitulation of Keap1/Cul3/Rbx1 complex-mediated Nrf2 degradation in HEK293 cells. B , Effect of dh404 on Keap1/Cul3/Rbx1 complex-mediated Nrf2 degradation in HEK293 cells. C , Effect of dh404 on assembling of Keap1/Cul3/Rbx1 complex in HEK293 cells. Cell lysates were subjected to immunoprecipitation (IP) and IB analysis using antibodies of Nrf2, Keap1, HA, Myc, and Gapdh as indicated. Results are representative of three independent experiments.

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Transfection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: HEK 293 cells were transfected with HA-Nrf2 and CBD-Keap1 as described in , and treated with dh404 (200 nM) in DMEM with 10% FBS as indicated. Immunoprecipitation (IP) and immunoblot (IB) analysis were performed using appropriate antibodies as indicated. Results are representative of three separated experiments. Cyo, cytosolic proteins; Nucle, nuclear proteins.

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: Sub-confluent cells were transfected with ARE-luc (200 ng) and pRL-TK (10 ng) for 24 hours, and then cultured with serum-free DMEM for another 24 hours. The quiescent cells were stimulated with dh404 as indicated, and then subjected to ARE reporter assays ( A ) as well as immunoblot analysis of cytosolic and nuclear Nrf2 expression ( B , left panel) or immunochemical staining of Nrf2 ( B , right panel).

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Transfection, Cell Culture, Western Blot, Expressing, Staining

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: A , Cells infected with 20 MOI of Ad-βGal, Ad-Nrf2, Ad-scramble or Ad-Nrf2 shRNA for 48 hours were stimulated with angiotensin II (Ang II, 0.5 µM) for 1 hour. B , Cells infected with 20 MOI of Ad-scramble or Ad-Nrf2 shRNA in the absence or presence of dh404 (200 nM) for 48 hours were stimulated with Ang II (0.5 µM) for 1 hour. C , Cells infected with 20 MOI of Ad-βGal, Ad-Nrf2, Ad-scramble or Ad-Nrf2 shRNA for 48 hours were stimulated with Ang II (2 µM) for 1 hour. D , Cells infected with 20 MOI of Ad-scramble or Ad-Nrf2 shRNA in the absence or presence of dh404 (200 nM) for 48 hours were stimulated with Ang II (2 µM) for 1 hour. O 2 .− and ONOO − formation was quantified as described in “ Materials and Methods ”. *p<0.05 vs control (−); #p<0.05 vs Ad-scramble Ang II (n = 8).

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Infection, shRNA, Control

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: A , Upper panel; left ventricular sections from mice (n = 3) treated with vehicle or dh404 (50 mg/kg) for 6 hours. Nrf2 expression and nuclear translocation were analyzed using confocal microscopy. Red is Nrf2; Green is cardiac myosin heavy chain; Blue is nuclei. Lower panel; semi-quantification of Nrf2 expression was performed by measuring integrated optical density (IOD)s of red signal of Nrf2 staining in 4 randomly chosen fields in each tissue section. Two sections from each mouse treated with or without dh404 were randomly chosen for analysis. B , Q-PCR analysis of dh404-induced expression of Nrf2 target genes in the heart. *p<0.05 vs vehicle, n = 3.

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Translocation Assay, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: Under normal conditions, a single Keap1 protein is able to target multiple Nrf2 proteins for destruction via ubiquitin-proteasome system, and only a small portion of Nrf2 proteins is translocated into the nuclus to activate Nrf2-driven transcription. Dh404 interacts with Cys-151 of Keap1 to inhibit the Keap1-dependent degradation of Nrf2. Thus, newly synthesized Nrf2 proteins saturate the capacity of Keap1 binding with Nrf2, accumulate in the cytoplasm and subsequently translocate into the nucleus, thereby facilitating Nrf2-driven transcription of antioxidant genes to protect against oxidative stress.

Article Snippet: H9C2 cells infected with Ad-Nrf2 or Ad-Nrf2 shRNA and rat neonatal cardiomyocytes treated with vehicle or dh404 (0.2 μM) for 1 hours were fixed as mentioned above, and stained with primary antibodies of Nrf2 (C-20, sc-722, Santa Cruz Biotechnology Inc.).

Techniques: Ubiquitin Proteomics, Synthesized, Binding Assay