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: Bioengineered

Article Title: Long noncoding RNA BBOX1-AS1 promotes the progression of gastric cancer by regulating the miR-361-3p/Mucin 13 signaling axis.

doi: 10.1080/21655979.2022.2072629

Figure Lengend Snippet: Figure 5. MUC13 was targeted by miR-361-3p. (a) MUC13 and CLDN4 were predicted and screened as the target genes of miR-361- 3p using GEPIA and ENCORI. (b) RNA pull-down analysis on the interaction between MUC13 and CLDN4 of miR-361-3p. **P < 0.001 vs Bio-NC. (c) The binding sites of MUC13 on miR-361-3p were predicted by StarBase. (d) The luciferase reporter analysis on the relationship between miR-361-3p and MUC13. **P < 0.001 vs miR-NC. (e, f) The expression level of MUC13 in GC clinical samples (e) and GC cell lines (f) was detected by qRT-PCR. (g) Pearson analysis revealed the expression relationship between miR-361-3p and MUC13.

Article Snippet: These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C.

Techniques: Binding Assay, Luciferase, Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: Long noncoding RNA BBOX1-AS1 promotes the progression of gastric cancer by regulating the miR-361-3p/Mucin 13 signaling axis.

doi: 10.1080/21655979.2022.2072629

Figure Lengend Snippet: Figure 6. MiR-361-3p knockdown promoted GC progression by targeting MUC13. (a) The expression level of miR-361-3p was evaluated in groups of si-NC, inhibitor-NC, inhibitor, si-MUC13, and si-MUC13+ inhibitor by qRT-PCR. (b-d) The cell proliferation (b), invasion (c), and apoptosis (d) were measured in groups of si-NC, inhibitor-NC, inhibitor, si-MUC13, and si-MUC13+ inhibitor using CCK-8, Transwell and flow cytometry assays, respectively. **P < 0.001 vs si-NC, &&P < 0.001 vs inhibitor-NC. ##P < 0.001 vs si-lnc +inhibitor.

Article Snippet: These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Nrf2 Transcriptional Activity Governs Intestine Development

doi: 10.3390/ijms23116175

Figure Lengend Snippet: Lack of transcriptional activity of Nrf2 changes colon morphology in 4-day-old pups. ( A ) Macroscopic changes in the colon length isolated from 4-day-old mice with similar body weight; brown debris in the Nrf2 tKO are remnants of indigested food that was unmovable from the gut despite extensive flushing. ( B ) Hematoxylin and eosin staining of the proximal and distal colon showing the disruption of the colon crypts and enlargement of the goblet cells. ( C ) The presence of enteroendocrine (ChrA) and goblet (Muc2) cells in the colon. ChrA (green), Muc2 (red), and nucleus (gray). N = 3 for the Nrf2 WT and Nrf2 tKO mice. ** p < 0.01; Representative images. Muc2—mucin 2, ChrA—chromogranin A. Mean ± SEM. Student’s t -test. Magnification 400× for B, scale bar 30 µm and magnification 250× for C, scale bar 45 µm.

Article Snippet: After washing in PBS, the samples were incubated overnight (4 °C) with mouse anti-Ki67 monoclonal IgG antibodies (dilution 1:500; Abcam), rabbit anti-Nrf2 IgG polyclonal antibodies (dilution 1:200; Proteintech), or rabbit anti-Notch1 IgG monoclonal antibodies (dilution 1:250; Cell Signaling) diluted in 3% GS in PBS with 0.05% Tween-20.

Techniques: Activity Assay, Isolation, Staining, Disruption

Journal: International Journal of Molecular Sciences

Article Title: Nrf2 Transcriptional Activity Governs Intestine Development

doi: 10.3390/ijms23116175

Figure Lengend Snippet: Significant histological abnormalities in the hindgut of Nrf2 tKO embryos. Hematoxylin and eosin staining of the intestine showed an enlargement of the epithelium in Nrf2 tKO fetuses at E14.5 (orange asterisk), earlier appearance of goblet cells at E15.5 (arrows), and further irregular organization and difference in the size of the goblet cells on days E17.5 and E18.5 (hash). N = 3–11 fetuses for the Nrf2 WT and Nrf2 tKO mice. Representative images, magnification 400×, scale bar 30 µm.

Article Snippet: After washing in PBS, the samples were incubated overnight (4 °C) with mouse anti-Ki67 monoclonal IgG antibodies (dilution 1:500; Abcam), rabbit anti-Nrf2 IgG polyclonal antibodies (dilution 1:200; Proteintech), or rabbit anti-Notch1 IgG monoclonal antibodies (dilution 1:250; Cell Signaling) diluted in 3% GS in PBS with 0.05% Tween-20.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Nrf2 Transcriptional Activity Governs Intestine Development

doi: 10.3390/ijms23116175

Figure Lengend Snippet: The Nrf2 transcriptional activity influences epithelial cell differentiation and the presence of enteroendocrine (ChrA) and goblet (Muc2) cells. ChrA (green), Muc2 (red), and nucleus (gray) expression in the female and male fetuses at selected gestation days; no sex-dependent changes. Green arrows—chromogranin A containing cells. N = 3–11 fetuses for the Nrf2 WT and Nrf2 tKO mice. Representative images, magnification 400×, scale bar 30 µm.

Article Snippet: After washing in PBS, the samples were incubated overnight (4 °C) with mouse anti-Ki67 monoclonal IgG antibodies (dilution 1:500; Abcam), rabbit anti-Nrf2 IgG polyclonal antibodies (dilution 1:200; Proteintech), or rabbit anti-Notch1 IgG monoclonal antibodies (dilution 1:250; Cell Signaling) diluted in 3% GS in PBS with 0.05% Tween-20.

Techniques: Activity Assay, Cell Differentiation, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Nrf2 Transcriptional Activity Governs Intestine Development

doi: 10.3390/ijms23116175

Figure Lengend Snippet: The Nrf2 expression changes in the embryo and hindgut during gestation. ( A ) The Nrf2 protein expression in embryos in the selected embryonic development days. ( B ) Nrf2 expression in female and male fetuses at selected gestation days. ( C ) Quantification of Nrf2 in the hindgut in Nrf2 WT fetuses; N = 6–11 fetuses of the Nrf2 WT mice. Mean ± SEM. One-way ANOVA. * p < 0.05, ** p < 0.01. Representative images, magnification 4×, scale bar 10 mm for A; magnification 400×, scale bar 30 µm for B.

Article Snippet: After washing in PBS, the samples were incubated overnight (4 °C) with mouse anti-Ki67 monoclonal IgG antibodies (dilution 1:500; Abcam), rabbit anti-Nrf2 IgG polyclonal antibodies (dilution 1:200; Proteintech), or rabbit anti-Notch1 IgG monoclonal antibodies (dilution 1:250; Cell Signaling) diluted in 3% GS in PBS with 0.05% Tween-20.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Nrf2 Transcriptional Activity Governs Intestine Development

doi: 10.3390/ijms23116175

Figure Lengend Snippet: Notch1 is reduced in the Nrf2 tKO embryos at the latest stages of development. ( A ) Notch1 expression in the female and male fetuses at selected gestation days. ( B ) Quantification of Notch1 in the hindgut of the Nrf2 WT and Nrf2 tKO fetuses. N = 3–11 fetuses for the Nrf2 WT and Nrf2 tKO mice. Mean ± SEM. Two-way ANOVA. ** p < 0.01. Representative images, magnification 200×, scale bar 50 µm.

Article Snippet: After washing in PBS, the samples were incubated overnight (4 °C) with mouse anti-Ki67 monoclonal IgG antibodies (dilution 1:500; Abcam), rabbit anti-Nrf2 IgG polyclonal antibodies (dilution 1:200; Proteintech), or rabbit anti-Notch1 IgG monoclonal antibodies (dilution 1:250; Cell Signaling) diluted in 3% GS in PBS with 0.05% Tween-20.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Nrf2 Transcriptional Activity Governs Intestine Development

doi: 10.3390/ijms23116175

Figure Lengend Snippet: The differential pattern of Ki67 expression under Nrf2 inhibition. ( A ) Ki67 expression in the female and male fetuses at selected gestation days. ( B ) Quantification of the Ki67 protein level changes in the hindgut in the Nrf2 WT and Nrf2 tKO fetuses. ( C ) Correlation between the mean changes in Nrf2 and Ki67 in the analyzed gestation days in the WT embryos. N = 3–11 fetuses in the Nrf2 WT and Nrf2 tKO mice. Mean ± SEM. Two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001. Representative images, magnification 400×, scale bar 30 µm.

Article Snippet: After washing in PBS, the samples were incubated overnight (4 °C) with mouse anti-Ki67 monoclonal IgG antibodies (dilution 1:500; Abcam), rabbit anti-Nrf2 IgG polyclonal antibodies (dilution 1:200; Proteintech), or rabbit anti-Notch1 IgG monoclonal antibodies (dilution 1:250; Cell Signaling) diluted in 3% GS in PBS with 0.05% Tween-20.

Techniques: Expressing, Inhibition

Journal: Redox Biology

Article Title: SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione

doi: 10.1016/j.redox.2021.102041

Figure Lengend Snippet: Nrf2, Membrane transporters, and GCLC in SARS-CoV2 infected VERO-E6 cells treated with Nelfinavir (Nel) or Remdesivir (Rem). Immunoblot of Nrf2 protein expression ( A , left panels) was assessed 6 hpi and 24 hpi, and by semi-quantitative fluorescence analysis 48 hpi ( A , right panels). Fluorophores were FITC (green) for Nrf2 protein labelling, DAPI (blue) for nuclei and Phalloidin-Alexa Fluor595 (orange) for the cytosolic space. Immunoblot of xCT and MRP1 membrane transport proteins ( B ), and GCLC protein ( C ) were carried out as described in the section Methods 24 hpi. Infection conditions and treatments with antivirals were the same of . Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals *p < 0.05, **p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used were: Nrf2 Polyclonal Antibody, FITC Conjugated (bs-1074R-FITC, Bioss Antibodies, 1:50); NQO1 (A180, Mouse mAb #3187, CST, 1:50); GST-pi (610,718, mouse mAb, BD Biosciences, 1:500); PE anti-human IL-6 Antibody (BioLegend, 1:1000); PE anti-human IL-10 Antibody (BioLegend, 1:1000).

Techniques: Infection, Western Blot, Expressing, Fluorescence

Journal: Molecular therapy. Nucleic acids

Article Title: TAZ ameliorates the microglia-mediated inflammatory response via the Nrf2-ROS-NF-κB pathway.

doi: 10.1016/j.omtn.2022.03.025

Figure Lengend Snippet: Figure 6. TAZ rescued microglia from apoptosis via Nrf2 (A and B) TAZ prevented microglia apoptosis via Nrf2. After introduction of the TAZ overexpression plasmid, cell apoptosis was evaluated in the absence or presence of the Nrf2 inhibitor ML385 by flow cytometry analysis or staining with Hoechst 33342, followed by visualization under a fluorescence microscope. N = 4. (C and D) TAZ attenuated Casp3 activity and reduced the levels of Casp3 and Bax mRNA along with recovery of Bcl2 expression through Nrf2. N = 5.

Article Snippet: After total and nuclear proteins were isolated and quantified using the kit, western blotting was performed with primary antibodies against TAZ (1:1,000, Thermo Fisher Scientific), phospho-TAZ (Ser89, 1:1,000, Cell Signaling Technology), Nrf2 (1:1,000, Proteintech), histone H3 (1:5,000, Proteintech), Gapdh (1:5,000, Proteintech), IkBa (1:1,000, Proteintech), phospho-IkBa (1:1,000, Cell Signaling Technology), andNF-kB subunit p65 (1:1,000, Cell Signaling Technology), as described previously.42 Measurement of antioxidant enzyme activity and GSH content After different treatments, proteins were extracted to assess the activity of the antioxidant enzymes SOD, CAT, GPX, and GR and resolve GSH content as well as the GSH/GSSG ratio in accordance with the corresponding assay kit (Beyotime).

Techniques: Over Expression, Plasmid Preparation, Cytometry, Staining, Microscopy, Activity Assay, Expressing

Journal: Molecular therapy. Nucleic acids

Article Title: TAZ ameliorates the microglia-mediated inflammatory response via the Nrf2-ROS-NF-κB pathway.

doi: 10.1016/j.omtn.2022.03.025

Figure Lengend Snippet: Figure 7. TAZ repressed NF-kB by enhancing antioxidant capacity dependent on Nrf2 (A) Western blot analysis of IkBa, phosphorylated IkBa, and p65 expression after transfection with the TAZ overexpression plasmid, followed by addition of the corresponding inhibitor for antioxidant enzymes, GSH synthesis, and Nrf2. p-IkBa, phosphorylated IkBa. N = 3. (B) Visualization of nc p65 after introduction of the TAZ overexpression plasmid and EGFP-p65 vector, followed by nc staining with Hoechst 33342 in the presence or absence of different inhibitors or the NF-kB activator BA. N = 3. (C) Determination of NF-kB transcriptional activity after introduction of the TAZ overexpression plasmid and pNFkB-luc vector in the presence or absence of different inhibitors or the NF-kB activator BA. N = 5. (D) Effects of the NF-kB activator BA on expression of IkBa, phosphorylated IkBa, and p65 after introduction of the TAZ overexpression plasmid. N = 3. (E) Activation of NF-kB by BA resisted the recruitment of TAZ on the levels for IL-1b, IL-6, and TNF-a mRNA. N = 5. (F) Activation of NF-kB by BA counteracted the rescue of TAZ on the release of IL-1b, IL-6 and TNF-a. N = 5.

Article Snippet: After total and nuclear proteins were isolated and quantified using the kit, western blotting was performed with primary antibodies against TAZ (1:1,000, Thermo Fisher Scientific), phospho-TAZ (Ser89, 1:1,000, Cell Signaling Technology), Nrf2 (1:1,000, Proteintech), histone H3 (1:5,000, Proteintech), Gapdh (1:5,000, Proteintech), IkBa (1:1,000, Proteintech), phospho-IkBa (1:1,000, Cell Signaling Technology), andNF-kB subunit p65 (1:1,000, Cell Signaling Technology), as described previously.42 Measurement of antioxidant enzyme activity and GSH content After different treatments, proteins were extracted to assess the activity of the antioxidant enzymes SOD, CAT, GPX, and GR and resolve GSH content as well as the GSH/GSSG ratio in accordance with the corresponding assay kit (Beyotime).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Staining, Activity Assay, Activation Assay

Journal: Molecular therapy. Nucleic acids

Article Title: TAZ ameliorates the microglia-mediated inflammatory response via the Nrf2-ROS-NF-κB pathway.

doi: 10.1016/j.omtn.2022.03.025

Figure Lengend Snippet: Figure 8. Schematic of TAZ regulation in the inflammatory response After shuttling into the nucleus, TAZ might drive transcription of Nrf2 by interacting with the TEAD transcription factor and induce nc translocation of Nrf2 to enhance antioxidant capacity with reduction of intracellular ROS, resulting in impediment of NF-kB activation to ameliorate the microglia-mediated inflammatory response. Simul- taneously, TAZ rescued microglia from apoptosis by preventing mitochondrial dysfunction, followed by blockage of mPTP opening and Cyt C release from mitochondria into the cytosol.

Article Snippet: After total and nuclear proteins were isolated and quantified using the kit, western blotting was performed with primary antibodies against TAZ (1:1,000, Thermo Fisher Scientific), phospho-TAZ (Ser89, 1:1,000, Cell Signaling Technology), Nrf2 (1:1,000, Proteintech), histone H3 (1:5,000, Proteintech), Gapdh (1:5,000, Proteintech), IkBa (1:1,000, Proteintech), phospho-IkBa (1:1,000, Cell Signaling Technology), andNF-kB subunit p65 (1:1,000, Cell Signaling Technology), as described previously.42 Measurement of antioxidant enzyme activity and GSH content After different treatments, proteins were extracted to assess the activity of the antioxidant enzymes SOD, CAT, GPX, and GR and resolve GSH content as well as the GSH/GSSG ratio in accordance with the corresponding assay kit (Beyotime).

Techniques: Translocation Assay, Activation Assay