gfp nrf2  (Addgene inc)

Journal: Antioxidants

Article Title: Activation of Nuclear Factor Erythroid 2-Related Factor 2 Transcriptionally Upregulates Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Expression and Inhibits Ectopic Calcification in Mice

doi: 10.3390/antiox13080896

Figure Lengend Snippet: Nrf2 activation augments ENPP1 expression. ( a ) Real-time RT-PCR analysis of ENPP1 on day 5. Gene expression was calibrated using the Rps18 housekeeping gene, and values are fold-changes compared to the control. Data are representative of three independent experiments performed in triplicate. * p < 0.05 vs. control, † p < 0.05 between samples. (b) Immunofluorescence analysis of ENPP1 on day 5. Cells cultured in medium with or without Nrf2 activator were subjected to immunofluorescence staining with an anti-ENPP1 primary antibody and fluorophore-labeled secondary antibody. Photographs were obtained using an exposure of 1.3 s for ENPP1 and 1/80 s for DAPI. (c) Signal intensity of ENPP1 analyzed using ImageJ (N = 8). Data are means ± SD and scatterplots with full dataset. * p < 0.05. (d) The ENPP1 mRNA level was augmented by Nrf2 overexpression. Gene expression 3 days after gene transfection was calibrated using the Rps18 housekeeping gene, and values are fold-changes compared to the control. Data are representative of three independent experiments performed in triplicate. Data are means ± SD and scatterplots with full dataset. * p < 0.05 vs. control. (e) Nrf2 activation augmented extracellular PPi. Concentration of extracellular PPi normalized to the protein concentration is indicated. Data are means ± SD and scatterplots with full dataset. (N = 3). * p < 0.05.

Article Snippet: The Nrf2 (accession number: NM_006164.5) expression plasmid (pcDNA3-EGFP-C4-Nrf2) [ ] was from Addgene (Watertown, MA, USA).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Gene Expression, Control, Immunofluorescence, Cell Culture, Staining, Labeling, Over Expression, Transfection, Concentration Assay, Protein Concentration

Journal: Antioxidants

Article Title: Activation of Nuclear Factor Erythroid 2-Related Factor 2 Transcriptionally Upregulates Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Expression and Inhibits Ectopic Calcification in Mice

doi: 10.3390/antiox13080896

Figure Lengend Snippet: In silico analysis of putative Nrf2-binding sites in the promoter region of ENPP1. The sequence of the ENPP1 promoter region (1500 bp upstream to 100 bp downstream of the start codon) was examined for putative Nrf2-binding sites using web-based software as described in the materials and methods section. Yellow highlighting indicates putative Nrf2-binding sites. Analyzed sequences are in red and numbered at left. The ChIP-qPCR primers are underlined. CDS, coding sequence (cyan).

Article Snippet: The Nrf2 (accession number: NM_006164.5) expression plasmid (pcDNA3-EGFP-C4-Nrf2) [ ] was from Addgene (Watertown, MA, USA).

Techniques: In Silico, Binding Assay, Sequencing, Software, ChIP-qPCR

Journal: Antioxidants

Article Title: Activation of Nuclear Factor Erythroid 2-Related Factor 2 Transcriptionally Upregulates Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Expression and Inhibits Ectopic Calcification in Mice

doi: 10.3390/antiox13080896

Figure Lengend Snippet: ChIP-qPCR analysis of the promoter region of ENPP1. Six sequences containing several putative Nrf2-binding sites shown in were analyzed by ChIP-qPCR. Cycle threshold (Ct) of percentage input DNA is shown. Samples immunoprecipitated using an anti-Nrf2 antibody from the control (middle open bar) and Nrf2 activation (right blue bar) groups were compared. Data are means ± SD and scatterplots with full dataset. (N = 3). * p < 0.05 between samples. NS, no significant difference.

Article Snippet: The Nrf2 (accession number: NM_006164.5) expression plasmid (pcDNA3-EGFP-C4-Nrf2) [ ] was from Addgene (Watertown, MA, USA).

Techniques: ChIP-qPCR, Binding Assay, Immunoprecipitation, Control, Activation Assay

Journal: Antioxidants

Article Title: Activation of Nuclear Factor Erythroid 2-Related Factor 2 Transcriptionally Upregulates Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Expression and Inhibits Ectopic Calcification in Mice

doi: 10.3390/antiox13080896

Figure Lengend Snippet: Nrf2 activation in ttw mice attenuated ectopic calcification. Representative 3D-rendered μCT images of control ttw mice ( a ) and ttw mice with Nrf2 activation ( b ). Blue arrow, ectopic calcification. Representative 2D μCT images of control ttw mice ( c ) and ttw mice with Nrf2 activation ( d ). Yellow arrowhead, ectopic calcification. ( e ) Ectopic calcification in ligament tissue between the second and third cervical vertebrae measured using ImageJ (n = 5 each). Data are means ± SD and scatterplots with full dataset. * p < 0.05 vs. control.

Article Snippet: The Nrf2 (accession number: NM_006164.5) expression plasmid (pcDNA3-EGFP-C4-Nrf2) [ ] was from Addgene (Watertown, MA, USA).

Techniques: Activation Assay, Control

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: Plasmids pcDNA3- EGFP- C4- Nrf2 (Addgene, 21549) (74), pCDNA3- Myc3- Nrf2 (Addgene, 21555) (74), pREP- 8xARE- GFPSV40- BFP (Addgene, 13,4910) (39), and HA- Ubiquitin (Addgene, 18,712) (75) were obtained from Addgene. p3xFlag- SSH1, p3xFlag- SSH1ΔC, p3xFlagSSH1ΔN, pEGFP- N1- p62- S403E, and pEGFP- N1- p62- S403A were previously generated by the Kang Lab (31, 38). p3xFlag- SSH1- CS was generated by the Kang lab during this study. p62- S349A- pEGFP- N1 (76) was kindly provided by Tanji Konikazu (Hirosaki University, Japan).

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: Plasmids pcDNA3- EGFP- C4- Nrf2 (Addgene, 21549) (74), pCDNA3- Myc3- Nrf2 (Addgene, 21555) (74), pREP- 8xARE- GFPSV40- BFP (Addgene, 13,4910) (39), and HA- Ubiquitin (Addgene, 18,712) (75) were obtained from Addgene. p3xFlag- SSH1, p3xFlag- SSH1ΔC, p3xFlagSSH1ΔN, pEGFP- N1- p62- S403E, and pEGFP- N1- p62- S403A were previously generated by the Kang Lab (31, 38). p3xFlag- SSH1- CS was generated by the Kang lab during this study. p62- S349A- pEGFP- N1 (76) was kindly provided by Tanji Konikazu (Hirosaki University, Japan).

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: Plasmids pcDNA3- EGFP- C4- Nrf2 (Addgene, 21549) (74), pCDNA3- Myc3- Nrf2 (Addgene, 21555) (74), pREP- 8xARE- GFPSV40- BFP (Addgene, 13,4910) (39), and HA- Ubiquitin (Addgene, 18,712) (75) were obtained from Addgene. p3xFlag- SSH1, p3xFlag- SSH1ΔC, p3xFlagSSH1ΔN, pEGFP- N1- p62- S403E, and pEGFP- N1- p62- S403A were previously generated by the Kang Lab (31, 38). p3xFlag- SSH1- CS was generated by the Kang lab during this study. p62- S349A- pEGFP- N1 (76) was kindly provided by Tanji Konikazu (Hirosaki University, Japan).

Techniques: Staining, Silver Staining, Two Tailed Test