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: 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: Food Science & Nutrition

Article Title: Trans‐Fatty Acids ( TFA ) Induced Vascular Injury Through the Regulation of the Sirt1‐Ppargc1a‐Nfe2l2 Signaling Pathway in Male Rats

doi: 10.1002/fsn3.70975

Figure Lengend Snippet: Effect of trans‐fatty acids on Sirt1/Ppargc1a/Nfe2l2 pathway of rat blood vessels. (A) Western blotting results of Silent information regulator 1 (Sirt1), Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (Ppargc1a), and nuclear factor erythroid 2‐related factor 2 (Nfe2l2). (B−D) Semi‐quantitative analyses of protein expression. Results are shown as mean ± SD ( n = 6). * p < 0.05 by contrast with ND group; # p < 0.05 by contrast with LTD group. ND, normal diet; LTD, 1% trans‐fatty acids diet; HTD, 4% trans‐fatty acids diet.

Article Snippet: The following primary antibodies were employed in the study: anti‐rabbit Bcl2 (Signalway Antibody, China), anti‐rabbit Bax (Cell Signaling Technology, USA), anti‐rabbit Ppargc1a (Affinity, China), anti‐rabbit Casp3 (GeneTex, USA), anti‐rabbit Cleaved Casp3 (Arigo Biolaboratories, China), anti‐rabbit Nfe2l2 (Medical & Biological Laboratories, China), anti‐rabbit Sirt1 (OriGene, USA), and anti‐rabbit β‐actin (Abcam, China).

Techniques: Western Blot, Expressing

Journal: Cells

Article Title: Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation

doi: 10.3390/cells8080794

Figure Lengend Snippet: LA induces Nrf2, which is likely dispensable for p53 degradation. ( A ) HCT116 cells were incubated with increasing doses of LA (125–1000 µM) for 48 h. Cells were thereafter subjected to SDS-PAGE and western blot analysis of p53, Nrf2 and its downstream target HO-1. EtOH (0 µM) served as solvent control. Hsp90 was visualized as loading control. ( B ) HCT116 cells were incubated for 48 h with LA in the presence or the absence of ML385, a pharmacological Nrf2 inhibitor. Hemin (200 µM, 24 h) was included as positive control for HO-1 induction. EtOH (0 µM) served as vehicle control. Cells were then lyzed and underwent western blot analysis of Nrf2, p53, HO-1, as well as p62. Hsp90 was used as loading control. ( C – F ) Densitometric quantification of NRF2 (C), p53 (D), HO-1 (E) and p62 (F) obtained from three independent experiments as described and shown in B. Data are given as mean + SEM ( n = 3). ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies included Hsp90α/β (F8, mouse monoclonal; Santa Cruz, no. sc-13119), p53 (DO-1, mouse monoclonal; Santa Cruz, no. sc-126), p53 (FL-393; rabbit polyclonal; Santa Cruz, no. sc-6243), p62 (mouse monoclonal; Santa Cruz, no. sc-28359), LC3B (rabbit monoclonal; Cell Signaling Technology, no. 3868), ATG5 (rabbit monoclonal, Cell Signaling Technology, no. 12994), ubiquitin (mouse monoclonal; Santa Cruz, no. sc-8017), Nrf2 antibody (rabbit monoclonal; GeneTex, no. GTX103322), MDM2 (mouse monoclonal; Santa Cruz, no. sc-56154), heme oxygenase-1 (HO-1; rabbit polyclonal; GeneTex, no. GTX101147), as well as p21 (C-19, rabbit polyclonal; Santa Cruz, no. sc-397).

Techniques: Incubation, SDS Page, Western Blot, Positive Control