Journal: Frontiers in Pharmacology

Article Title: The essential oil from the rhizomes of Stahlianthus involucratus attenuates the progression of vascular aging and atherosclerosis by regulating Nrf2-mediated mitochondrial quality

doi: 10.3389/fphar.2025.1579333

Figure Lengend Snippet: Impact of EOSIR on the Nrf2 signaling pathway in AS mice. (A) WB assays for Keap1, Nrf2, NQO1, and HO-1 expression. (B) qRT-PCR assays of Keap1, Nrf2, NQO1, and HO-1 mRNA expression. (C) Immunofluorescence staining of Nrf2 in mouse aortic tissue ( x ¯ ±SD, n = 3) ( * P < 0.05 or ** P < 0.01 vs. control group; # P < 0.05 or ## P < 0.01 vs. HFD group).

Article Snippet: ML385 (an Nrf2 inhibitor), and Bardoxolone Methyl (BARD) (an Nrf2 agonist) were obtained from MCE (HY-100523, SMB00376-10MG, New Jersey, United States).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Control

Journal: Frontiers in Pharmacology

Article Title: The essential oil from the rhizomes of Stahlianthus involucratus attenuates the progression of vascular aging and atherosclerosis by regulating Nrf2-mediated mitochondrial quality

doi: 10.3389/fphar.2025.1579333

Figure Lengend Snippet: Effect of EOSIR on ox-LDL-induced Nrf2 signaling activation in HUVECs. (A) WB assays for Keap1, Nrf2, and NQO1 expression following Nrf2 inhibition. (B) WB assays for Keap1, Nrf2, and NQO1 expression after Nrf2 silencing. (C) Immunofluorescence staining of ox-LD-induced Nrf2 nuclear translocation in HUVECs. ( x ¯ ±SD, n = 3) ( * P < 0.05 or ** P < 0.01 or *** P < 0.001 vs. control group; # P < 0.05 or ## P < 0.01 vs. ox-LDL group; △ P < 0.05 or △△ P < 0.01 vs. EOSIR group; ▲ P < 0.05 or ▲▲ P < 0.01 vs. si-Nrf2 group).

Article Snippet: ML385 (an Nrf2 inhibitor), and Bardoxolone Methyl (BARD) (an Nrf2 agonist) were obtained from MCE (HY-100523, SMB00376-10MG, New Jersey, United States).

Techniques: Activation Assay, Expressing, Inhibition, Immunofluorescence, Staining, Translocation Assay, Control

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Positive correlation between FGFR4 and NRF2 in gastric cancer. A) Pearson's correlation analysis between FGFR4 mRNA level and single-sample gene set enrichment analysis (ssGSEA) scores for NRF2 targets' signature in the TCGA cohort. B) GSEA was performed using samples with FGFR4 high expression compared to samples with FGFR4 low expression in TCGA cohort. NRF2 signature was significantly enriched in FGFR4 high expression samples. C) Immunofluorescence analysis shows an increase of NRF2 nuclear staining (green) and FGFR4 expression (red) in TFF1-KO mouse neoplastic gastric tissues, as compared to normal gastric tissues from the TFF1-WT mice (scale of 10 μm is shown in the merged image). The arrows point to nuclei. The right panels show bright field image of H&E staining. D) Western blot analysis demonstrates an increase of p-FGFR4 (Y642), FGFR4, NRF2 and HO1 protein levels in neoplastic gastric tissues (KO), as compared to normal tissue samples (WT) from mice. RT-qPCR analysis of Fgfr4, Fgf15 and Ho1 mRNA expression in gastric tissues of TFF1-KO mouse as compared to TFF1-WT. *P < 0.05, **P < 0.01 and ***P < 0.001 is considered significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: FGFR4 knockdown reduces NRF2 protein and transcription activity levels. A) Western blot shows an increase of FGFR4, NRF2 and HO1 proteins after H. pylori infection (3h) as compared to uninfected negative control (NC). FGFR4 siRNA knockdown abrogated this increase. B) Immunofluorescence assay demonstrates an increase of NRF2 nuclear staining in H. pylori Infected cells. This increase was abolished with FGFR4 siRNA knockdown. C) Quantification of nuclear NRF2-positive staining in at least 200 cells from three images is presented as a percentage in the right panel. Data are graphed with mean ± SEM. D) The NRF2 transcriptional activity was measured by the ARE luciferase reporter assay following infection with H. pylori in MKN28 cells. H. pylori induced the activity of the reporter, whereas FGFR4 siRNA knockdown reversed this effect. The luciferase reporter activity values were normalized to β-gal expression levels and are represented as luciferase activity relative to control. E-F) RT-qPCR of FGF19 (E) and HO1 (F) following infection with H. pylori (3h) in MKN28 cells transfected with control siRNA or FGFR4 siRNA. ***P < 0.001.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Knockdown, Activity Assay, Western Blot, Infection, Negative Control, Immunofluorescence, Staining, Luciferase, Reporter Assay, Expressing, Control, Quantitative RT-PCR, Transfection

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: NRF2 nuclear expression is associated with H. pylori infection and tumorigenesis in mouse gastric tissues. A) Immunofluorescence analysis shows an increase of NRF2 (green) nuclear staining (arrow heads) and FGFR4 expression (red; arrow heads) in wild-type mice (C57/B6) gastric tissues after H. pylori ( PMSS1 strain ) infection for four weeks (scale of 10 μm is shown in the merged image). The right panels show bright field images of H&E staining. The arrowheads point to the nuclei. B) Western blot analysis demonstrates an increase in the protein levels of p-FGFR4 (Y642), FGFR4, NRF2 and HO1 in gastric tissues of mice infected with PMSS1 as compared to uninfected mice. C) RT-qPCR analysis of Fgfr4, Fgf15 and Ho1 mRNA expression in gastric tissues of mice infected with PMSS1 as compared to uninfected mice. *P < 0.05, **P < 0.01, ***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: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Expressing, Infection, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: FGFR4 inhibition decreases NRF2 in vitro and in vivo . A) Western blot analysis in MKN28 cells. Treatment with FGFR4 inhibitor (H3B-6527) abrogates H. pylori (7.13)-mediated increase in NRF2. B) The ARE luciferase reporter assay was used as a measure of NRF2 transcriptional activity under similar conditions as in B, demonstrating a significant induction of luciferase activity by H. pylori (7.13) infection, an effect that was abolished with the H3B-6527 inhibitor. The luciferase reporter activity values were normalized to β-gal expression levels and are represented as luciferase activity relative to control. C) Immunofluorescence analysis shows an increase of NRF2 (green) nuclear staining and FGFR4 expression (red) in wild-type mice (C57/B6) gastric tissues after four weeks of infection with H. pylori (PMSS1); nuclear localization of NRF2 was abolished after treatment with H3B-6527 (H3B); arrows point to nuclei. The right panels show bright field images of H&E staining. D) Western blot analysis of mouse gastric tissues shows an increase of p-FGFR4 (Y642), FGFR4 and NRF2 in infected mice; this increase was abolished after treatment with H3B-6527. E) RT-qPCR analysis of Fgfr4 and Ho1 mRNA expression in gastric tissues of mice infected with PMSS1 and treated or not with H3B-6527 (H3B) and compared to uninfected mice. *P < 0.05, **P < 0.01, ***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: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Inhibition, In Vitro, In Vivo, Western Blot, Luciferase, Reporter Assay, Activity Assay, Infection, Expressing, Control, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: FGFR4 induces P62 to promote NRF2 protein stability. A) Western blot analysis using MKN28 cells uninfected or infected with H. pylori (7.13, 3h) . Infected cells show high levels of p-FGFR4 (Y642), FGFR4, NRF2, p-P62 (S349) and P62. These changes were reversed upon FGFR4 siRNA knockdown. The relative intensity ratios of NRF2/β-Actin, p-P62/β-Actin, and P62/β-Actin were calculated by ImageJ software and shown on the right panel. The results are expressed as mean ± SEM of at least three independent experiments. B) Western blot analysis following treatment of MKN28 cells with FGF19 (30 min). This stimulation led to increases in p-FGFR4 (Y642), FGFR4, NRF2, p-P62 (S349) and P62. These changes were reversed with FGFR4 siRNA knockdown. The Bar graphs show the relative intensity ratios of NRF2/β-Actin, p-P62/β-Actin, and P62/β-Actin were calculated and shown on the right panel. The results are expressed as mean ± SEM of at least three independent experiments. C-D) Western blot showing cycloheximide chase assay of NRF2 using FGFR4 siRNA (C) or P62 siRNA (D) at the indicated time points, following H. pylori infection (3h). NRF2 protein stability was reduced following FGFR4 or P62 knockdown. The half-life time (t1/2) of NRF2 was calculated using GraphPad Prism software and plotted on the right side of panels C and D, respectively. *P < 0.05, **P < 0.001.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Western Blot, Infection, Knockdown, Software

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: FGFR4, P62, and KEAP1 coexist in the same protein complex. A-B) Immunoprecipitation and western blot analysis following P62 pulldown (A) or FGFR4 pulldown (B) using MKN28 cells infected with H. pylori 7.13 (3 h). Immunoprecipitations and their corresponding input samples were subjected to immunoblotting with P62 FGFR4 Keap1 and NRF2 antibodies. The infection was confirmed using CagA antibody and equal amounts of protein loading were confirmed in the input samples using GAPDH antibody. C) Proximity ligation assay was performed in MKN28 cells transfected with control or FGFR4 siRNA and infected with H. pylori ( 7.13). The presence of red signals indicates positive ligation and proximity of the proteins, indicative of interaction. Using FGFR4 and P62 antibodies, the results indicated the presence of FGFR4-P62 interaction (red signals) following H. pylori infection (left upper panel). This interaction was not detected with FGFR4 siRNA (middle upper panel). The lower panels display the negative control for PLA background reaction. Control cells were transfected with Ctrl siRNA, infected with H. pylori and probed with a single antibody against FGFR4 (lower left panel) or P62 (lower right panel). The upper right panel displays a negative control for the PLA background with no antibody. Maximum intensity projection is presented on the right and lower sides of each image. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Immunoprecipitation, Western Blot, Infection, Proximity Ligation Assay, Transfection, Control, Ligation, Negative Control

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: FGFR4 promotes cell expansion and correlates with NRF2 in gastric cancer. A) Representative images of spheroids showing an increase in size and number of spheroids after H. pylori infection, an effect that was abrogated upon FGFR4 knockdown (scale 100 μm). B & C) Quantification of the size of at least twenty spheroids (B) and sphere counts (C) from three different fields. D) Western blot analysis using spheroids derived from MKN28 cells uninfected or infected with H. pylori (7.13). Infected cells show higher levels of FGFR4, NRF2, p-P62 (S349) and P62 than uninfected cells. These changes were reversed with FGFR4 siRNA knockdown. E) representative images of immunohistochemistry staining of FGFR4 and NRF2 in serial tissue sections from coded human gastric tissues with normal histology (NG), high grade dysplasia (HGD) and cancer. Original magnification 10× and 40X are shown on the left side of the panel). A progressive increase in FGFR4 and NRF2 was observed along different histological pathologies (HGD and cancer) as compared to NG. F-G) The graph bars summarize the immunohistochemical staining index scores according to the number of cases in percentage for each histological stage on the gastric tissue microarrays. We used Cochran-Mantel-Haenszel Statistical analysis of disease to analyze IHC scores across histological stages, FGFR4 ( X 2 = 41.28, Df = 2, P < 0.0001 ) and NRF2 ( X 2 = 12.3, Df = 2, P < 0.01 ). H) Spearman's Correlation analysis shows a positive correlation between FGFR4 and NRF2 index scores in gastric cancer tissues.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Infection, Knockdown, Western Blot, Derivative Assay, Immunohistochemistry, Staining, Immunohistochemical staining

Journal: Redox Biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: A schematic diagram illustrating the role of FGFR4 in activating and stabilizing NRF2. Exposure of cells to H. pylori infection induces oxidative stress and FGFR4 expression. FGFR4 binds to P62. The FGFR4-P62 complex binds to KEAP1 in NRF2 degradation complex, competing with binding of KEAP1 top NRF2. NRF2 escapes degradation, accumulates, and translocates to the nucleus to induce transcription of antioxidant response genes.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Infection, Expressing, Binding Assay

Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: Smurf1 increases the binding affinity between p62 and Keap1 to activate Nrf2. A LN229 cells were transfected with HA or HA-Smurf1. Cell lysates were subjected to western blot analysis with the indicated antibodies (anti-p62, anti-p-p62 S349 , anti-p-p62 S403 , anti-HA, and anti-β-actin). Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin (mean ± SD from 3 independent experiments). Values were normalized against the intensity of LN229 transfected with HA. * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t -test. B LN229 cells treated by p62 siRNA oligos were transfected with Flag or Flag-Smurf1, then overexpressed GFP, GFP-p62 WT , GFP-p62 S349A , or GFP-p62 K7A/D69A . Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p-p62 S349 , anti-GFP, and anti-β-actin). C LN229 cells transfected with Flag or Flag-Smurf1 were treated with DMSO or Rapamycin (100 nM, 24 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p-p62 S349 , anti-p62, anti-p-mTOR, anti-mTOR, anti-p-p70S6K, anti-p70S6K, anti-Flag and anti-β-actin). D LN229 cells treated by p62 siRNA oligos were transfected with Flag or Flag-Smurf1, then overexpressed GFP, GFP-p62 WT , GFP-p62 S349A or GFP-p62 K7A/D69A . Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of NQO1 mRNA in LN229 transfected with GFP, Flag plasmids and p62 siRNA oligos; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. E 293T cells overexpressed GFP-p62 were transfected HA, HA-Smurf1, si-Control, or si-Smurf1. Cell lysates were immunoprecipitated by anti-Keap1 antibody. The immunoprecipitates and the input were probed with anti-GFP, anti-Keap1, anti-Nrf2 and anti-HA antibodies. F 293T cells overexpressed GFP-p62 S349A were transfected HA, HA-Smurf1, or si-Smurf1. Cell lysates were immunoprecipitated by anti-Keap1 antibody. The immunoprecipitates and the input were probed with anti-GFP, anti-Keap1, anti-Nrf2 and anti-HA antibodies. G LN229 cells treated with control or Nrf2 siRNA oligos were transfected with Flag or Flag-Smurf1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of p62, NQO1, and HO1 mRNA. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos and Flag; mean ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. H LN229 cells treated with control, p62, or Nrf2 siRNA oligos were transfected with Flag or Flag-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies. I Smurf1 promotes the phosphorylation of p62 S349 by activating mTORC1 signalling pathway. p62 S349 phosphorylation enhance p62 binding affinity with Keap1 and Nrf2 nuclear translocation. The activated Nrf2 partially participates to the transcriptional activation of p62

Article Snippet: The primary antibodies were used: Smurf1 (Santa, sc100616; Abcam, ab57573), Keap1 (Proteintech, 10503-2-AP), Keap1 (Abcam, ab118285), p62 (Enzo, BML-PW9860), p62 (MBL, PM045), p62 (MBL, M162-3), LC3B (Sigma, L7543), LC3B (Novus, NB100-2220), NBR1 (Proteintech, 160004-1-AP), HA (MBL, M180-3), Nrf2 (Proteintech, 16396-1-AP), Flag (Sigma, F1804).

Techniques: Binding Assay, Transfection, Western Blot, Two Tailed Test, Control, Immunoprecipitation, Phospho-proteomics, Translocation Assay, Activation Assay

Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: NBR1 enhances Smurf1-drived Nrf2-mediated oxidative stress response. A LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Smurf1, anti-NBR1, anti-p62, and anti-β-actin). B LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1 and NBR1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (mean ± SD from 3 independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. C LN229 cells were fixed after treating with control (PBS) or H 2 O 2 (200 µM, 2 h), and then immunofluorescence stained with anti-Smurf1, anti-p62, and anti-NBR1 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). ** p < 0.01 as determined by unpaired two-tailed Student’s t -test. D LN229 cells were transfected with control or Smurf1 siRNA oligos. Thereafter, the cells were divided into three groups: (i) cultured in regular medium, (ii) treated with H 2 O 2 (200 µM, 2 h), and (iii) after treating with H 2 O 2 (200 µM, 2 h), cultured in regular medium for another 12 h. Cell lysates were prepared and subjected to western blot analysis with indicated antibodies (anti-NBR1, anti-p62, anti-p-p62 S349 , anti-p-p62 S403 , anti-Smurf1, and anti-β-actin). E LN229 cells transfected with control or Smurf1 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1, p62, NBR1, and NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (mean ± SD from 3 independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. F LN229 cells treated with control or Smurf1 siRNA oligos and fixed after treating with control (PBS) or H 2 O 2 (200 µM, 2 h), and then immunofluorescence stained with anti-NBR1, anti-Smurf1 and anti-p62 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. G LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H 2 O 2 (200 µM, 2 h) after overexpressing HA or HA-NBR1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-Flag, and anti-β-actin). H LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H 2 O 2 (200 µM, 2 h) after overexpressing HA or HA-NBR1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos and Flag; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. I Role of Smurf1 in stress response. Stress upregulates the level of Smurf1, leading to the increased formation and material exchange of p62 liquid droplets. Smurf1 promotes the phosphorylation of p62 S349 by activating mTORC1 signalling pathway. These further promote the transcription of anti-stress proteins by competitively binding Keap1 and mediating Nrf2 nuclear import. The activated Nrf2 increases the mRNA level of Smurf1, p62, and NBR1 to promote the formation and enlarging of p62 liquid droplets as positive feedback

Article Snippet: The primary antibodies were used: Smurf1 (Santa, sc100616; Abcam, ab57573), Keap1 (Proteintech, 10503-2-AP), Keap1 (Abcam, ab118285), p62 (Enzo, BML-PW9860), p62 (MBL, PM045), p62 (MBL, M162-3), LC3B (Sigma, L7543), LC3B (Novus, NB100-2220), NBR1 (Proteintech, 160004-1-AP), HA (MBL, M180-3), Nrf2 (Proteintech, 16396-1-AP), Flag (Sigma, F1804).

Techniques: Transfection, Control, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Cell Culture, Phospho-proteomics, Binding Assay

Journal: Drug Design, Development and Therapy

Article Title: Bu Shen Huo Xue Formula Provides Neuroprotection Against Spinal Cord Injury by Inhibits Oxidative Stress by Activating the Nrf2 Signaling Pathway

doi: 10.2147/dddt.s487307

Figure Lengend Snippet: Figure 4 BSHXF reduced apoptosis after spinal cord injury. (A)TUNEL staining to assess the cell apoptosis rate. (B) Quantification of Tunel-positive cells in each group. (C) Protein expression levels of Bax and Bcl2 were measured by Western blot analysis. (D) Quantification of relative Bcl2/Bax protein levels. (E) Quantitative analysis of the number of C-caspase3+ cells obtained from three regions of interest. (F) Immunofluorescent images of the apoptosis marker C-caspase3 (red) in the damaged area on day 14 post-injury, with nuclei counterstained with DAPI (blue). All experiments were performed in triplicate, and data are presented as means ± SEM, n = 5 per group. **p < 0.01.

Article Snippet: Anti-GFAP (BA0056) and anti-MAP2 (BM1243) antibodies were acquired from Boster Biological Engineering Co.; anti-MBP (NBP2-22121) and anti-GAP43 (NBP1-92713) antibodies were sourced from NOVUS; and antibodies targeting Bax (2772), cleaved-caspase-3 (C-caspase3)(9664), Nrf2 (12721), and GAPDH (9664) were obtained from Cell Signaling Technology.

Techniques: TUNEL Assay, Staining, Expressing, Western Blot, Marker

Journal: Drug Design, Development and Therapy

Article Title: Bu Shen Huo Xue Formula Provides Neuroprotection Against Spinal Cord Injury by Inhibits Oxidative Stress by Activating the Nrf2 Signaling Pathway

doi: 10.2147/dddt.s487307

Figure Lengend Snippet: Figure 9 BSHXF mitigated LPS-induced apoptosis and axonal degeneration in neurons. (A and B) Fluorescence images showing the localization of Bcl2 and C-caspase3. (C) Quantification of fluorescence intensity for Bcl2 and C-caspase3. (D) Western blot analysis of C-caspase3, Bcl2, and Bax protein expression. (E) Quantification of the bands from Western blot analysis. (F) Mitochondrial membrane potential levels detected by JC-1 staining. (G) Ratios of polymeric (red) and monomeric (green) forms of JC-1, reflecting MMP in the indicated groups. (H) MAP2 visualized by fluorescein isothiocyanate fluorescence (red), with nuclei counterstained by DAPI (blue). (I) Statistical results of MAP2 fluorescence intensity across the groups. Data are presented as means ± SEM. *p < 0.05 and **p < 0.01 compared with the Con group; #p < 0.05 and ##p < 0.01 compared with the LPS group.

Article Snippet: Anti-GFAP (BA0056) and anti-MAP2 (BM1243) antibodies were acquired from Boster Biological Engineering Co.; anti-MBP (NBP2-22121) and anti-GAP43 (NBP1-92713) antibodies were sourced from NOVUS; and antibodies targeting Bax (2772), cleaved-caspase-3 (C-caspase3)(9664), Nrf2 (12721), and GAPDH (9664) were obtained from Cell Signaling Technology.

Techniques: Fluorescence, Western Blot, Expressing, Membrane, Staining

Journal: Drug Design, Development and Therapy

Article Title: Bu Shen Huo Xue Formula Provides Neuroprotection Against Spinal Cord Injury by Inhibits Oxidative Stress by Activating the Nrf2 Signaling Pathway

doi: 10.2147/dddt.s487307

Figure Lengend Snippet: Figure 10 BSHXF reduced neuronal damage caused by oxidative stress by activating the Nrf2 pathway. (A) Western blot analysis of the expression of Nrf2 pathway-related proteins, including (Nrf2,NQO1, and HO-1). (B) Quantification of the bands from the Western blot analysis. (C) Western blot analysis of apoptotic proteins, including C-caspase3, Bcl2, and Bax. (D) Quantification of the bands from the Western blot analysis. (E) Immunofluorescence images showing Bcl2 expression in neurons. (F) Immunofluorescence images showing C-caspase3 expression in neurons. (G) Immunofluorescence images showing MAP2 expression in neurons. (H) Statistical chart of fluorescence signals. Data are presented as means ± SEM. #p < 0.05 and ##p < 0.01 compared with the LPS group; &p < 0.05 and &&p < 0.01 compared with the BSHXF group.

Article Snippet: Anti-GFAP (BA0056) and anti-MAP2 (BM1243) antibodies were acquired from Boster Biological Engineering Co.; anti-MBP (NBP2-22121) and anti-GAP43 (NBP1-92713) antibodies were sourced from NOVUS; and antibodies targeting Bax (2772), cleaved-caspase-3 (C-caspase3)(9664), Nrf2 (12721), and GAPDH (9664) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Chinese medicine

Article Title: The SIRT1/Nrf2 signaling pathway mediates the anti-pulmonary fibrosis effect of liquiritigenin.

doi: 10.1186/s13020-024-00886-1

Figure Lengend Snippet: Fig. 4 Liquiritigenin activates Nrf2 and reverses TGF-β1-induced oxidative stress. A H2DCFCDA probe represented ROS levels. Scale bars represent 100 μm. B MitoSOX probe represented mitochondrial ROS levels. Scale bars represent 100 μm. C Immunofluorescence microscopy observed the effect of liquiritigenin on Nrf2 nuclear translocation, with scale bars representing 50 μm. D–E The impact of liquiritigenin on Nrf2 nuclear translocation was assessed through Western blotting. F–G mRNA levels of HO-1 and NQO-1 analyzed by qPCR. H–K Protein expression levels of CAT, HO-1, and NQO-1 determined by Western blotting. Data are presented as means ± standard deviation, and each experiment was independently repeated at least three times. (*P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Next, the membranes were incubated with primary antibodies overnight at 4 °C, including β-actin monoclonal antibody (1:5000, Proteintech, China), α-SMA monoclonal antibody (1:1000, CST, USA), collagen I polyclonal antibody (1:1000, Millipore, USA), SIRT1 polyclonal antibody (1:1000, Proteintech, China), CAT polyclonal antibody (1:3000, Abcam, UK), HO-1 polyclonal antibody (1:1000, Abcam, UK), NQO-1 monoclonal antibody (1:1000, Abcam, UK), Nrf2 polyclonal antibody (1:1000, Proteintech, China), smad3 monoclonal antibody (1:1000, Proteintech, China), Pho-smad3 monoclonal antibody (1:1000, Proteintech, China), smad4 monoclonal antibody (1:1000, Proteintech, China), Pho-smad4 monoclonal antibody (1:1000, Proteintech, China).

Techniques: Immunofluorescence, Microscopy, Translocation Assay, Western Blot, Expressing, Standard Deviation

Journal: Chinese medicine

Article Title: The SIRT1/Nrf2 signaling pathway mediates the anti-pulmonary fibrosis effect of liquiritigenin.

doi: 10.1186/s13020-024-00886-1

Figure Lengend Snippet: Fig. 8 The SIRT1/Nrf2 signaling pathway mediates the effects of liquiritigenin on bleomycin-induced pulmonary fibrosis in mice. A To assess whether EX527 and ML385 influence the effects of liquiritigenin on pulmonary fibrosis induced by bleomycin, mice were orally administered with 100 mg/kg of liquiritigenin 3 h before receiving EX527 and ML385 from day 15 to day 28 following intratracheal injection of bleomycin. B Lung morphology and ECM deposition were evaluated through HE and Masson staining. Scale bars represent 100 μm. C The survival curve of mice was recorded. D Scoring of pulmonary fibrosis severity in mice. E Hydroxyproline content, an indicator of fibrosis, was quantified using biochemical methods. F–G mRNA levels of myofibroblast markers, collagen I and α-SMA, were analyzed through qPCR. H–J Protein expression levels of collagen I and α-SMA were determined by Western blotting. Data are presented as means ± standard deviation, and all experiments were independently repeated at least three times. (*P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Next, the membranes were incubated with primary antibodies overnight at 4 °C, including β-actin monoclonal antibody (1:5000, Proteintech, China), α-SMA monoclonal antibody (1:1000, CST, USA), collagen I polyclonal antibody (1:1000, Millipore, USA), SIRT1 polyclonal antibody (1:1000, Proteintech, China), CAT polyclonal antibody (1:3000, Abcam, UK), HO-1 polyclonal antibody (1:1000, Abcam, UK), NQO-1 monoclonal antibody (1:1000, Abcam, UK), Nrf2 polyclonal antibody (1:1000, Proteintech, China), smad3 monoclonal antibody (1:1000, Proteintech, China), Pho-smad3 monoclonal antibody (1:1000, Proteintech, China), smad4 monoclonal antibody (1:1000, Proteintech, China), Pho-smad4 monoclonal antibody (1:1000, Proteintech, China).

Techniques: Injection, Staining, Expressing, Western Blot, Standard Deviation

Journal: Chinese medicine

Article Title: The SIRT1/Nrf2 signaling pathway mediates the anti-pulmonary fibrosis effect of liquiritigenin.

doi: 10.1186/s13020-024-00886-1

Figure Lengend Snippet: Fig. 9 The SIRT1/Nrf2 signaling pathway mediates the effects of liquiritigenin on bleomycin-induced pulmonary oxidative stress in mice. A DHE staining reflects lung ROS levels. Scale bars represent 100 μm. B–D Biochemical quantification of MDA and GSH levels, as well as SOD activity. E, F qPCR analysis of HO-1 and NQO-1 mRNA levels. G–J Western blotting determined the protein expression levels of CAT, HO-1, and NQO-1. Data are presented as means ± standard deviation, and all experiments were independently repeated at least three times. (*P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Next, the membranes were incubated with primary antibodies overnight at 4 °C, including β-actin monoclonal antibody (1:5000, Proteintech, China), α-SMA monoclonal antibody (1:1000, CST, USA), collagen I polyclonal antibody (1:1000, Millipore, USA), SIRT1 polyclonal antibody (1:1000, Proteintech, China), CAT polyclonal antibody (1:3000, Abcam, UK), HO-1 polyclonal antibody (1:1000, Abcam, UK), NQO-1 monoclonal antibody (1:1000, Abcam, UK), Nrf2 polyclonal antibody (1:1000, Proteintech, China), smad3 monoclonal antibody (1:1000, Proteintech, China), Pho-smad3 monoclonal antibody (1:1000, Proteintech, China), smad4 monoclonal antibody (1:1000, Proteintech, China), Pho-smad4 monoclonal antibody (1:1000, Proteintech, China).

Techniques: Staining, Activity Assay, Western Blot, Expressing, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

doi: 10.1074/jbc.m112.381509

Figure Lengend Snippet: FIGURE 1. Depletion of KLF2 fails to inhibit laminar flow-induced Nrf2-dependent gene expression. A, HUVECs transfected with control, Nrf2, or KLF2 siRNA (100 pM) for 3 days were exposed to atheroprotective flow (12 dynes/cm2) for 16–24 h. Protein expression of HO-1, NQO1, Nrf2, eNOS, and tubulin was determinedbyimmunoblottingwithspecificantibodies.B–F,mRNAexpressionofKLF2,eNOS,Nrf2,HO-1,andferritinheavychain(FerritinHC)wasdetermined by qRT-PCR analysis as follows. Total RNA was isolated by using TRIzol reagent (Invitrogen), and reverse transcription reaction was conducted by using TaqMan reverse transcription reagents (Applied Biosystems) following the manufacturer’s instructions. Quantitative real-time PCR was conducted with 1 l of template cDNA using Power SYBR Green (Applied Biosystems) as described under “Experimental Procedures.” Data are expressed as mean S.D. from three independent experiments (n 2–3). **, p 0.01; NS, not significant.

Article Snippet: Cells were stained with monoclonal anti-mouse ERK5 antibody (Cell Signaling Technology) and rabbit polyclonal Nrf2 antibody (H300; Santa Cruz Biotechnology) at 4 °C overnight.

Techniques: Gene Expression, Transfection, Control, Expressing, Quantitative RT-PCR, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Journal of Biological Chemistry

Article Title: Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

doi: 10.1074/jbc.m112.381509

Figure Lengend Snippet: FIGURE 2. Inhibition of ERK5 suppresses laminar flow-mediated Nrf2-de- pendent gene expression. A, HUVECs transfected with either control or ERK5 siRNA for 3 days were exposed to atheroprotective flow (12 dynes/cm2). Protein expression of HO-1, eNOS, pERK5, ERK5, and -actin was determined by immunoblotting with specific antibodies. B, HUVECs were exposed to atheroprotective flow for 16–24 h in the presence of either DMSO or BIX02189 (10 M). Protein expression of HO-1, NQO1, eNOS, pERK5, ERK5, and tubulin was determined by immunoblotting with specific antibodies. Data are representative from three independent experiments.

Article Snippet: Cells were stained with monoclonal anti-mouse ERK5 antibody (Cell Signaling Technology) and rabbit polyclonal Nrf2 antibody (H300; Santa Cruz Biotechnology) at 4 °C overnight.

Techniques: Inhibition, Gene Expression, Transfection, Control, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

doi: 10.1074/jbc.m112.381509

Figure Lengend Snippet: FIGURE 3. ERK5 activation increases transcriptional activity of Nrf2. A, HUVECs were co-transfected with either control or ERK5 siRNA (si-ERK5) (100 pM) and pGL3-ARE-luciferase (ARE-luc) for 2 days. Cells were then exposed to atheroprotective flow for 16–24 h, and luciferase activity was measured by using the Dual-Luciferase reporter assay kit (Promega) and GloMax 20/20 luminometer (Promega). Transfection efficiency was normalized by Renilla luciferase activity derived from pRL-TK construct. B, cells were co-transfected with pBIND-Nrf2, pG5-luciferase, and pERK5 in the presence or absence of pCA-MEK5 as indicated in the figure. Luciferase activity was measured by using Dual-Luciferase reporter assay kit (Promega) and GloMax 20/20 lumi- nometer (Promega). Transfection efficiency was normalized by Renilla lucif- erase activity derived from pRL-TK construct. Data are expressed as mean S.D. from three independent experiments. *, p 0.05; **, p 0.01.

Article Snippet: Cells were stained with monoclonal anti-mouse ERK5 antibody (Cell Signaling Technology) and rabbit polyclonal Nrf2 antibody (H300; Santa Cruz Biotechnology) at 4 °C overnight.

Techniques: Activation Assay, Activity Assay, Transfection, Control, Luciferase, Reporter Assay, Derivative Assay, Construct

Journal: Journal of Biological Chemistry

Article Title: Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

doi: 10.1074/jbc.m112.381509

Figure Lengend Snippet: FIGURE 4. ERK5 is required for laminar flow-induced Nrf2 nuclear trans- location. A, HUVECs were pretreated with BIX02189 (10 M) for 16–24 h and then exposed to laminar flow (12 dynes/cm2) or static condition. Both cyto- solic and nuclear compartments were obtained by cellular fractionation as described under “Experimental Procedures.” Cellular localization of Nrf2 and ERK5 was determined by immunoblotting with anti-Nrf2 and anti-ERK5 anti- bodies, respectively, and proper fractionation of cytosolic and nuclear com- partments was confirmed by specific antibodies against tubulin and lamin B, respectively. B, HEK293 cells were infected with adenovirus LacZ or CA-MEK5for40h,andcellularlocalizationofNrf2wasdeterminedbyimmu- noblotting with anti-Nrf2 antibody. Data are representative of three inde- pendent experiments with similar results.

Article Snippet: Cells were stained with monoclonal anti-mouse ERK5 antibody (Cell Signaling Technology) and rabbit polyclonal Nrf2 antibody (H300; Santa Cruz Biotechnology) at 4 °C overnight.

Techniques: Cell Fractionation, Western Blot, Fractionation, Infection

Journal: Journal of Biological Chemistry

Article Title: Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

doi: 10.1074/jbc.m112.381509

Figure Lengend Snippet: FIGURE 5. ERK5 binds to Nrf2 in a kinase activity-dependent manner. A, cells were co-transfected with pMyc-Nrf2 and pXpress-ERK5 in the presence or absence of pHA-CA-MEK5. Cell lysates were used for immunoprecipitation (IP) analysis with -Myc antibody 24 h after transfection and then blotted with anti-Xpress and anti-Nrf2 antibodies as indicated in the figure. The total amount of ERK5 and CA-MEK5 from whole cell lysates was determined by immuno- blotting (IB). B, HUVECs were exposed to laminar flow for the indicated times or static condition. Cell lysates were then immunoprecipitated with anti-ERK5 antibody followedbyimmunoblottingwithanti-Nrf2antibody.TheexpressionofERK5andNrf2wasdeterminedbyimmunoblottingwithspecificantibodies.Asteriskindicates nonspecificband.ON,overnight.C,HUVECswereexposedtolaminarflowfortheindicatedtimesorstaticcondition,andinvivoprotein-proteininteractionofERK5and Nrf2wasdeterminedusingtheinvivofluorescenceprotein-proteininteractiondetectionkit(Duolink,Olink)followingthemanufacturer’sinstructions.Flow-induced ERK5-Nrf2interactionwasdetectedasreddots.Cellswerestainedwithanti-ERK5antibodyandanti-Nrf2antibody,andnucleuswasstainedwithDAPI(200).D,cells were transfected with various pACT-ERK5 fragments as indicated in the figure, and cell lysates were then incubated with recombinant GST-Nrf2 followed by immu- noblottingwithanti-VP16antibody(leftpanel).TheexpressionofvariousERK5fragmentsfromwholecelllysateswasdeterminedbyimmunoblottingwithanti-VP16 antibody. Data are representative of three independent experiments. Right panel, schematic model showing the length and legion of each fragments of ERK5 (frag-A to frag-D). P, phosphorylation. E, cells were transfected with pERK5-WT or pERK5-AEF in the presence or absence of pCA-MEK5, and cell lysates were then incubated with recombinant GST-Nrf2. The binding affinity of ERK5 to GST-Nrf2 was determined by immunoblotting with anti-ERK5 antibody (top panel). The equal amount of GST-Nrf2 was verified in Coomassie Blue staining (middle panel). The expression of ERK5 from whole cell lysates was determined by immunoblotting with anti-ERK5 antibody (bottom panel). Data are representative of three independent experiments.

Article Snippet: Cells were stained with monoclonal anti-mouse ERK5 antibody (Cell Signaling Technology) and rabbit polyclonal Nrf2 antibody (H300; Santa Cruz Biotechnology) at 4 °C overnight.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Incubation, Recombinant, Phospho-proteomics, Binding Assay, Western Blot, Staining, Expressing

Journal: Journal of Biological Chemistry

Article Title: Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

doi: 10.1074/jbc.m112.381509

Figure Lengend Snippet: FIGURE 6. ERK5 activation protects against oxidative stress-induced cytotoxicity dependent on Nrf2. A, HUVECs transfected with either control or ERK5 siRNA (siERK5, 100 pM) for 3 days were exposed to laminar flow overnight and then exposed to 100 M H2O2 for 8 h to induce oxidative stress. Cell viability was determined by MTT analysis as described under “Experimental Procedures.” B–D, HUVECs transfected with either control or Nrf2 siRNA (100 pM) were infected with either Ad-CA-MEK5 or Ad-LacZ for 2 days as indicated and then exposed to 100 M H2O2 for 8 h. Cell viability and apoptosis were determined by MTT analysis (B), TUNEL staining (C), and Western blotting assay (D). TUNEL-positive cells were observed under the fluorescence microscope. The numbers of TUNEL-positive cells are presented by bar graph. Cell lysates were used for immunoblotting with anti-PARP1, anti-caspase 3 (Casp3) (against for cleaved form of caspase 3), and anti-tubulin antibodies. Data are expressed as mean S.D. from three independent experiments. **, p 0.01; NS, not significant.

Article Snippet: Cells were stained with monoclonal anti-mouse ERK5 antibody (Cell Signaling Technology) and rabbit polyclonal Nrf2 antibody (H300; Santa Cruz Biotechnology) at 4 °C overnight.

Techniques: Activation Assay, Transfection, Control, Infection, TUNEL Assay, Staining, Western Blot, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

doi: 10.1074/jbc.m112.381509

Figure Lengend Snippet: FIGURE 7. Inhibition of ERK5 suppresses Nrf2 nuclear translocation in mouse aorta in vivo. Nrf2 nuclear translocation in mouse aortic endothelium was analyzed by en face immunofluorescence staining assay using anti-Nrf2 antibody (red). Mice were treated with either 10 mg/kg of BIX02189 (in 25% DMSO) or vehicle control (same volume of 25% DMSO) by intraperitoneal injection. Endothelium of thoracic aorta was stained with anti-vascular endothelial-cadherin (VE-cadherin) antibody for endothelial cell-cell junction staining (green), Topro3 for nuclear staining (blue), and anti-Nrf2 antibody (red) and photographed under a confocal microscope.

Article Snippet: Cells were stained with monoclonal anti-mouse ERK5 antibody (Cell Signaling Technology) and rabbit polyclonal Nrf2 antibody (H300; Santa Cruz Biotechnology) at 4 °C overnight.

Techniques: Inhibition, Translocation Assay, In Vivo, Immunofluorescence, Staining, Control, Injection, Microscopy

Journal: PLoS ONE

Article Title: Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

doi: 10.1371/journal.pone.0141622

Figure Lengend Snippet: β-actin was monitored as the internal standard to ensure similar gel loading of the starting materials in each sample. Blot images were cropped for comparison (E). Electroacupuncture treatment significantly increased the levels of phospho-Akt protein, HO-1 protein, Nrf2 total and nucleoprotein compared with group L or group SEL. In wortmannin-treated rabbits, phosphorylation of Akt was significantly decreased while HO-1 protein, Nrf2 total and nucleoprotein was slightly decreased compared with group EL. All values were expressed as mean ±SD (n = 10; *P<0 . 05; **P<0 . 01 , using one-way ANOVA and the Bonferroni test for multiple comparisons). The blots were representative of three independent experiments.

Article Snippet: After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK).

Techniques: Comparison, Phospho-proteomics

Journal: PLoS ONE

Article Title: Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

doi: 10.1371/journal.pone.0141622

Figure Lengend Snippet: (original magnification×400): (A) The pictures of immunofluorescence staining. Green stands for Nrf2-FITC stained sections, while blue stands for images of DAPI stained nuclei. The overlay color of blue staining in nucleus, accompanied with green staining both in cytoplasm and nucleus was considered to be positive. (B) Quantification of nuclear localization of Nrf2 protein among six groups. Electroacupuncture protocols dramatically increased translocation of Nrf2 from cytoplasm into nucleus compared with group L or SEL. To some degree, pretreatment with wortmannin counteracted nuclear accumulation of Nrf2 protein, while wortmannin alone had no effects. Data were representative of three independent experiments. Values were expressed as mean ± SD (n = 10; **P<0 . 01 , using one-way ANOVA and the Bonferroni test for multiple comparisons).

Article Snippet: After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK).

Techniques: Immunofluorescence, Staining, Translocation Assay

Journal: Journal of Inflammation Research

Article Title: Sodium Propionate Enhances Nrf2-Mediated Protective Defense Against Oxidative Stress and Inflammation in Lipopolysaccharide-Induced Neonatal Mice

doi: 10.2147/JIR.S303105

Figure Lengend Snippet: Primers for Real-Time Quantitative PCR Analysis in Humans

Article Snippet: For Western blotting analysis, rabbit monoclonal antibody against Nrf2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Journal of Inflammation Research

Article Title: Sodium Propionate Enhances Nrf2-Mediated Protective Defense Against Oxidative Stress and Inflammation in Lipopolysaccharide-Induced Neonatal Mice

doi: 10.2147/JIR.S303105

Figure Lengend Snippet: SP treatment dampens inflammation and oxidative stress, amplifies Nrf2 activation and vascularization in LPS-treated neonatal mice lung. When the newborn mice were at day of life 6, they received intraperitoneal injection of 1 mg/kg LPS, whereas the control mice received an equal volume injection of sterile saline solution. The LPS group were then randomly divided into two groups that received vehicle or sodium propionate (1.2 mg/g) for 7 d. ( A ) Effect of SP on lung and heart organ index. ( B ) Effect of SP on the mRNA expressions of inflammatory cytokines. ( C and D ) Effect of SP on serum and lung SOD activity, respectively. ( E ) Effect of SP on the mRNA expressions of SOD1, SOD2, Gclm and Txn . ( F ) Representative images of Western blots. ( G ) The protein levels of Keap-1, Nrf2 and vWF. ( H ) Immunohistochemistry for vWF in the lung section. * P <0.05 vs saline; ** P <0.01 vs saline; *** P <0.001 vs saline; # P <0.05 vs LPS; ## P <0.01 vs LPS; ### P <0.001 vs LPS. Values are mean±SE, n=6 per group.

Article Snippet: For Western blotting analysis, rabbit monoclonal antibody against Nrf2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Injection, Control, Sterility, Saline, Activity Assay, Western Blot, Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Sodium Propionate Enhances Nrf2-Mediated Protective Defense Against Oxidative Stress and Inflammation in Lipopolysaccharide-Induced Neonatal Mice

doi: 10.2147/JIR.S303105

Figure Lengend Snippet: SP prevents LPS-evoked pulmonary alveolar remodeling depending on Nrf2. When the newborn mice (WT and Nrf2 -/- ) were at day of life 6, they received intraperitoneal injection of 1 mg/kg LPS, whereas the control mice received an equal volume injection of sterile saline solution. The LPS group were then randomly divided into two groups that received vehicle or sodium propionate (1.2 mg/g) for 7 d. ( A ) The protein expression of Nrf2. ( B ) Representative H&E stained lung sections (magnification, ×200). ( C ) Quantification of MLI. ( D ) Lung and heart organ index. ( E ) Serum SOD activity. ( F ) The mRNA levels of inflammatory cytokines and Nrf2 target genes. * P <0.05 vs saline; ** P <0.01 vs saline; # P <0.05 vs LPS; ## P <0.01 vs LPS; n.s. vs no significance. Values are mean±SE, n=6 per group.

Article Snippet: For Western blotting analysis, rabbit monoclonal antibody against Nrf2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Injection, Control, Sterility, Saline, Expressing, Staining, Activity Assay

Journal: Journal of Inflammation Research

Article Title: Sodium Propionate Enhances Nrf2-Mediated Protective Defense Against Oxidative Stress and Inflammation in Lipopolysaccharide-Induced Neonatal Mice

doi: 10.2147/JIR.S303105

Figure Lengend Snippet: SP blocks LPS-induced oxidative stress in HPMECs. HPMECs were stimulated with LPS (1 μg/mL) for 2 h, followed by treated with SP (0.6 mM) for 24 h. ( A ) Intracellular ROS was determined using DHE staining. ( B ) Relative fluorescent intensity of DHE staining. ( C ) The mRNA levels of antioxidant genes. ( D ) Representative Western Blots and quantification of Keap-1 and Nrf2. ( E ) Representative images of immunofluorescence staining of Nrf2 (green). Nuclei were stained with DAPI (blue). * P <0.05 vs PBS; ** P <0.01 vs PBS, *** P <0.001 vs PBS; # P <0.05 vs LPS; ## P <0.01 vs LPS. Values are mean±SE, n=3 per group.

Article Snippet: For Western blotting analysis, rabbit monoclonal antibody against Nrf2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Staining, Western Blot, Immunofluorescence

Journal: Journal of Inflammation Research

Article Title: Sodium Propionate Enhances Nrf2-Mediated Protective Defense Against Oxidative Stress and Inflammation in Lipopolysaccharide-Induced Neonatal Mice

doi: 10.2147/JIR.S303105

Figure Lengend Snippet: SP blocks LPS-triggered HPMECs injury depending on Nrf2 activation. HPMECs were stimulated with ML358 (5 μM) for 1 h, followed by stimulated with LPS (1 μg/mL) for 2 h and treated with SP (0.6 mM) for additional 24 h. ( A ) mRNA levels of inflammatory cytokines. ( B ) mRNA levels of antioxidant genes. ( C ) The mRNA levels of angiogenesis-related genes. ( D ) Relative fluorescent intensity of DHE staining. ( E ) Representative DHE staining images. # P <0.05 vs LPS; ## P <0.01 vs LPS; ### P <0.001 vs LPS; † P <0.05 vs LPS + SP; †† P <0.01 vs LPS + SP; n.s. vs no significance. Values are mean ± SE, n=3 per group.

Article Snippet: For Western blotting analysis, rabbit monoclonal antibody against Nrf2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Quercetin-3-O-β-D-glucuronide attenuates osteoarthritis by inhibiting cartilage extracellular matrix degradation and inflammation

doi: 10.1016/j.jot.2024.01.007

Figure Lengend Snippet: The effect of Q3GA on the Nrf2 pathway in IL-1β-induced chondrocytes. (A) The 3D structure of Q3GA. (B) The 3D structure of the Nrf2 protein. (C) Molecular docking results of Q3GA with potential candidate proteins. (D) A combined 3D structure of Q3GA and Nrf2. (E) Western blots revealed the protein expression of Nrf2 in the nucleus and HO-1 in whole cells, n = 3. (F) Immunofluorescence assay was used to detect the expression of Nrf2 (scale bar: 50 μm). # P < 0.05, ## P < 0.01, and ### P < 0.001, compared with the IL-1β-induced group.

Article Snippet: Primary antibodies against ACAN (ab36861) and ADAMTS5 (ab41037) were purchased from Abcam (Cambridge, UK); iNOS (18985-1-AP), COX2 (12375-1-AP), p65 (66535-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), and LaminB1 (12987-1-AP) from Proteintech (Wuhan, China); p-p65 (WL02169) from Wanleibio (Shenyang, China); Col2a1(AF0135) and MMP13 (AF5355), MMP3 (AF0217), IκBα (AF5002) and GAPDH (AF7021) from Affinity (Melbourne, USA).

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: Journal of Orthopaedic Translation

Article Title: Quercetin-3-O-β-D-glucuronide attenuates osteoarthritis by inhibiting cartilage extracellular matrix degradation and inflammation

doi: 10.1016/j.jot.2024.01.007

Figure Lengend Snippet: The effect of knockdown of Nrf2 on IL-1β-induced chondrocytes. (A) The mRNA expression of Nrf2 and Ho-1 in chondrocytes treated with or without Nrf2 siRNA was detected using qRT–PCR, n = 3. (B) The protein expression of Nrf2 and HO-1 in chondrocytes treated with or without Nrf2 siRNA was detected using western blots, n = 3. (C) The protein expression of p-p65 and p65 in chondrocytes was detected using western blots, n = 3. (D) The protein expression of ACAN, ADAMTS5, MMP13, MMP3, and iNOS was detected using western blots, n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Primary antibodies against ACAN (ab36861) and ADAMTS5 (ab41037) were purchased from Abcam (Cambridge, UK); iNOS (18985-1-AP), COX2 (12375-1-AP), p65 (66535-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), and LaminB1 (12987-1-AP) from Proteintech (Wuhan, China); p-p65 (WL02169) from Wanleibio (Shenyang, China); Col2a1(AF0135) and MMP13 (AF5355), MMP3 (AF0217), IκBα (AF5002) and GAPDH (AF7021) from Affinity (Melbourne, USA).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: Quercetin-3-O-β-D-glucuronide attenuates osteoarthritis by inhibiting cartilage extracellular matrix degradation and inflammation

doi: 10.1016/j.jot.2024.01.007

Figure Lengend Snippet: Q3GA inhibits ECM degradation in OA rats. (A) Immunohistochemical staining assay for ACAN, ADAMTS5, MMP13, and Nrf2 in the cartilage. (B) Quantitative analysis of ACAN, ADAMTS5, MMP13, and Nrf2 using immunohistochemistry staining, (scale bar: 100 μm). *** P < 0.001, compared with the control group; # P < 0.05, ## P < 0.01 and ### P < 0.001, compared with the OA group.

Article Snippet: Primary antibodies against ACAN (ab36861) and ADAMTS5 (ab41037) were purchased from Abcam (Cambridge, UK); iNOS (18985-1-AP), COX2 (12375-1-AP), p65 (66535-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), and LaminB1 (12987-1-AP) from Proteintech (Wuhan, China); p-p65 (WL02169) from Wanleibio (Shenyang, China); Col2a1(AF0135) and MMP13 (AF5355), MMP3 (AF0217), IκBα (AF5002) and GAPDH (AF7021) from Affinity (Melbourne, USA).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium

doi: 10.1016/j.jcmgh.2022.09.004

Figure Lengend Snippet: Esophageal dysplasia in Keap1-cKO mice was abolished by Nrf2 deletion. ( A ) Immunohistochemical staining for KEAP1, NRF2, and NQO1 at 1 week after Tam administration. In KEAP1-deleted cells, NRF2 accumulated in the nucleus, and NQO1 was expressed at high levels. Scale bars: 50 μm. ( B ) NQO1 immunohistochemistry and HE staining in Keap1 floxB/floxB :: Nrf2 –/– and K5CreERT2 :: Keap1 floxB/floxB :: Nrf2 –/– mouse esophagi. Scale bars: 100 μm.

Article Snippet: NRF2 (D9J1B) , Rat monoclonal , Cell Signaling Technology , 14596 , 1:400 , —.

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium

doi: 10.1016/j.jcmgh.2022.09.004

Figure Lengend Snippet: NRF2 activation decreases 4 weeks after Tam treatment. ( A ) Immunohistochemical staining for NQO1. Population of NQO1-positive cells decreased over time in groups treated with each dose. Scale bars: 400 μm. ( B ) Quantification of NQO1-positive cells in the esophageal epithelium of mice treated with the 3 doses. Percentage of NQO1-positive cells was calculated as the NQO1-positive area within the total esophageal epithelial area in each mouse (n = 7–12 mice per group). ( C ) Positive correlations of percentage of NQO1-positive cells with thicknesses of keratinous and epithelial layers 1 week after administration of 3 doses of Tam (n = 29 mice). Line shows the two-tailed Pearson correlation. ( D ) Nqo1 and Gclc mRNA expression levels. Gene expression was decreased throughout the 4-week period in each group (n = 4–6 mice per group). Data are presented as means ± standard deviations. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001 according to Welch t test.

Article Snippet: NRF2 (D9J1B) , Rat monoclonal , Cell Signaling Technology , 14596 , 1:400 , —.

Techniques: Activation Assay, Immunohistochemical staining, Staining, Two Tailed Test, Expressing, Gene Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium

doi: 10.1016/j.jcmgh.2022.09.004

Figure Lengend Snippet: NRF2-activated cells are selectively eliminated and induce DNA damage in neighboring KEAP1-normal cells. ( A ) NQO1-positive floating cells were present in the Keap1-cKO mouse esophagus. ( B ) Immunofluorescence staining for NQO1 and COL17A1. White arrows indicate COL17A1-weak areas. NRF2-activated cells exhibited weak COL17A1 staining. ( C ) COL17A1-weak areas were counted in each section of esophageal epithelium from Keap1-cKO mice (n = 9). COL17A1-weak areas were particularly abundant in NQO1-positive basal cells. ( D ) Immunofluorescence staining for NQO1 and Loricrin. ( E ) Schematic of the esophageal epithelium of Keap1-cKO mice. NQO1-positive cells exhibited weaker COL17A1 staining than KEAP1-normal cells. Scale bars: 50 μm. Data are presented as means ± standard deviations. ∗∗ P < .01, as compared using Welch t test.

Article Snippet: NRF2 (D9J1B) , Rat monoclonal , Cell Signaling Technology , 14596 , 1:400 , —.

Techniques: Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium

doi: 10.1016/j.jcmgh.2022.09.004

Figure Lengend Snippet: NRF2-activated cells are selectively eliminated and induce DNA damage in neighboring KEAP1-normal cells. ( A ) Immunofluorescence staining for NQO1 and Ki67. The number of Ki67-positive cells was increased, especially among NRF2-activated cells, in the esophageal epithelium of Keap1-cKO mice. ( B ) Percentages of Ki67-positive cells among NQO1-negative and NQO1-positive cells in the Keap1-cKO mouse esophagus (n = 9). ( C ) The number of Ki67-positive cells in the basal layer was normalized to the length of the basement membrane (n = 12 control mice and n = 9 Keap1-cKO mice). ( D ) Ki67 mRNA expression level (n = 9 control mice and n = 10 Keap1-cKO mice). ( E ) Immunofluorescence staining for NQO1 and γH2A.X. The number of γH2A.X-positive cells was significantly increased, especially among NRF2-activated cells, in the Keap1-cKO mouse esophagus. ( F ) Percentages of γH2A.X-positive cells among NQO1-negative and NQO1-positive cells in the Keap1-cKO mouse esophagus. ( G ) Immunohistochemical staining for γH2A.X. ( H ) The number of γH2A.X-positive cells in the basal layer was normalized to the length of the basement membrane (n = 4–11 mice per group). Scale bars: 50 μm. Data are presented as means ± standard deviations. ∗ P < .05 and ∗∗∗ P < .001, as compared using Welch t test.

Article Snippet: NRF2 (D9J1B) , Rat monoclonal , Cell Signaling Technology , 14596 , 1:400 , —.

Techniques: Immunofluorescence, Staining, Membrane, Control, Expressing, Immunohistochemical staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium

doi: 10.1016/j.jcmgh.2022.09.004

Figure Lengend Snippet: Additional NRF2 activation in Keap1-cKO::Nrf2 SA mice promotes elimination of NRF2-activated cells. ( A ) Immunohistochemical staining for NQO1. NQO1-positive cells were distributed segmentally in the esophageal epithelium of both Keap1-cKO::Nrf2 SA mice and Keap1-cKO mice. Scale bars: 400 μm. ( B ) Quantification of esophageal epithelial length. The epithelial length was greatest in Keap1-cKO::Nrf2 SA mice (n = 9–12 mice per group). ( C ) Quantification of NQO1-positive cells in the esophageal epithelium. The number of NQO1-positive cells decreased earlier in Keap1-cKO::Nrf2 SA mouse esophagus than in Keap1-cKO mouse esophagus (n = 8–10 mice per group). ( D and E ) Immunohistochemical staining for Ki67. A greater increase in number of Ki67-positive cells was observed in the Keap1-cKO::Nrf2 SA mouse esophagus than in the Keap1-cKO mouse esophagus. Scale bars: 50 μm. ( F and G ) Immunohistochemical staining for γH2A.X. Greater increase in number of γH2A.X-positive cells was observed in Keap1-cKO::Nrf2 SA mouse esophagus than in Keap1-cKO mouse esophagus. Scale bars: 50 μm. Data are presented as means ± standard deviations. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001 according to Welch t test.

Article Snippet: NRF2 (D9J1B) , Rat monoclonal , Cell Signaling Technology , 14596 , 1:400 , —.

Techniques: Activation Assay, Immunohistochemical staining, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium

doi: 10.1016/j.jcmgh.2022.09.004

Figure Lengend Snippet: Accelerated proliferation of KEAP1-normal cells. ( A ) UMAP visualization of cells isolated from control and Keap1-cKO mouse esophagi. Left panel , 4853 cells are colored on basis of cell type. Right panel , 2472 and 2381 cells are colored on basis of their origin from control and Keap1-cKO mice, respectively. ( B ) Nqo1 expression in epithelial cells. ( C ) GSEA enrichment plot comparing Nqo1 Low and Nqo1 High epithelial cells using the NRF2 pathway gene set obtained from Wiki Pathways. ( D ) GSEA enrichment plot comparing Nqo1 Low and Nqo1 High epithelial cells using hallmark gene sets. Left panel shows the gene sets enriched in Nqo1 High cells. Only the reactive oxygen species pathway was significantly enriched among the hallmark gene sets. The right two panels show the hallmark gene sets enriched in Nqo1 Low cells. ( E ) GSEA enrichment plot comparing control and Nqo1 Low epithelial cells. The gene sets E2F targets and G2/M checkpoint were significantly enriched in Nqo1 Low cells.

Article Snippet: NRF2 (D9J1B) , Rat monoclonal , Cell Signaling Technology , 14596 , 1:400 , —.

Techniques: Isolation, Control, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium

doi: 10.1016/j.jcmgh.2022.09.004

Figure Lengend Snippet: Antibodies Used for Immunohistochemistry and Immunofluorescence

Article Snippet: NRF2 (D9J1B) , Rat monoclonal , Cell Signaling Technology , 14596 , 1:400 , —.

Techniques: Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3 *

doi: 10.1074/jbc.M114.571984

Figure Lengend Snippet: XBP1u protein was essential for disturbed flow-induced HDAC3 up-regulation. A , VEGF-PI3K/Akt pathway was involved in disturbed flow (4 h)-induced up-regulation of XBP1 expression and splicing. DM , DMSO (vehicle control); SU , SU5416 (1 μmol/liter, VEGF receptor inhibitor); PD , PD98059 (5 μmol/liter, ERK inhibitor); LY , LY294002 (5 μmol/liter, PI3K/Akt inhibitor). B , knockdown of XBP1 or IRE1 α abolished disturbed flow (4 h)-induced HDAC3 up-regulation. UT , untransfected; NT , non-target shRNA transfected; Xsh , XBP1 shRNA transfected; Ish , IRE1 α shRNA transfected. Disturbed flow was applied to HUVECs 72 h post transfection for 4 h. C , Overexpression of spliced XBP1 down-regulated HDAC3 protein. FLAG indicates the exogenous XBP1s protein. D , spliced XBP1 suppressed HDAC3 gene transcription. RLA , relative luciferase activity. pGL3-luc basic vector was included as negative control, whereas grp78-Luc vector was used as positive control. Mock, pShuttle- LacZ plasmid; XBP1s, pShuttle-FLAG- XBP1s plasmid; XBP1u , pShuttle-FLAG- XBP1u plasmid. E , XBP1u antagonized XBP1s on the regulation of HDAC3 protein. HUVECs were co-infected with Ad- XBP1s and Ad- XBP1u at 10 MOI each for 48 h. Ad-null was included as control and to compensate the MOI. FLAG indicates the exogenous XBP1s and XBP1u proteins. F , XBP1u and XBP1s differentially bound to HDAC3 promoter in response to disturbed flow. ChIP assay was performed to analyze the binding of XBP1u and XBP1s to the HDAC3 promoter in static and disturbed flow-treated HUVECs (4 h). Six sets of primer pairs covered the +1 ∼ −1467 region ( upper panel ), and PCR showed that XBP1u and XBP1s differentially bound to −960 ∼ −1195 region in response to disturbed flow ( lower panel ). SS , shear stress. Data presented are representative or average of three independent experiments. *, p < 0.05.

Article Snippet: Human Nrf2 siRNA (sc-37030) was purchased from Santa Cruz Biotechnology and reconstituted accordingly.

Techniques: Expressing, Control, Knockdown, shRNA, Transfection, Over Expression, Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Positive Control, Infection, Binding Assay, Shear

Journal: The Journal of Biological Chemistry

Article Title: Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3 *

doi: 10.1074/jbc.M114.571984

Figure Lengend Snippet: XBP1u-mediated cell survival was through regulation of HO-1 expression. A , HO-1 inhibitor SnPPIX abolished the protective effect of XBP1u overexpression on cell survival under H 2 O 2 challenging. B , quantitative RT-PCR revealed that over-expression of XBP1u or HDAC3 up-regulated HMOX-1 mRNA level without effect on Nrf2 mRNA level. C , Western blot analysis showed that overexpression of XBP1u or HDAC3 up-regulated both Nrf2 and HO-1 protein levels. D , knockdown of Nrf2 abolished XBP1u or HDAC3-induced HO-1 expression. CTLsi , control siRNA. E , overexpression of XBP1u or HDAC3 increased Nrf2 nuclear translocation and HO-1 expression in the infected and adjacent cells. Double immunofluorescence staining was performed with anti-Akt1 ( red ) and anti-Nrf2 ( green ) antibodies or with anti-FLAG ( red ) for exogenous XBP1u or HDAC3 and anti-HO-1 ( green ) antibodies. Data presented are representatives or average from three independent experiments. *, p < 0.05.

Article Snippet: Human Nrf2 siRNA (sc-37030) was purchased from Santa Cruz Biotechnology and reconstituted accordingly.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Knockdown, Control, Translocation Assay, Infection, Double Immunofluorescence Staining

Journal: The Journal of Biological Chemistry

Article Title: Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3 *

doi: 10.1074/jbc.M114.571984

Figure Lengend Snippet: XBP1 was essential for disturbed flow-induced up-regulation of HO-1. A , overexpression of XBP1u up-regulated HO-1 and Akt phosphorylation in a dose-dependent manner. HUVECs were infected with Ad- XBP1u at MOI indicated for 24 h, followed by Western blot analysis. Ad-null was included to compensate the MOI. B , overexpression of XBP1u maintained a high level of Akt1 phosphorylation and HO-1 expression. HUVECs were infected with Ad- XBP1u at 10 MOI for 24 h and 48 h, followed by Western blot analysis. Ad-null was included as control. FLAG indicates the exogenous XBP1u. C , knockdown of XBP1 via shRNA lentivirus ( XBP1sh ) decreased basal level of Akt1 phosphorylation and HO-1 expression. Non-target shRNA lentivirus ( NTsh ) was included as control. D , knockdown of XBP1 via shRNA lentivirus ( XBP1sh ) abolished disturbed flow-induced HO-1 expression. Non-target shRNA lentivirus ( NTsh ) was included as control. E , Nrf2 was necessary for flow-induced HO-1 expression. HUVECs were transfected with control siRNA ( CTLsi ) or Nrf2 siRNA ( Nrf2si ) for 72 h, followed by disturbed flow for 4 h. F , flow stabilized Nrf2 via post-translational modification. HUVECs were treated with 1 μmol/liter actinomycin D ( AD ) or 30 mg/liter cycloheximide ( CH ) for 1 h, followed by disturbed flow for 4 h or kept at static conditions in the presence of the inhibitors. DMSO ( DM ) was included as vehicle control. G , AZD2014 abolished Ad-XBP1u ( X1u ) or Ad-HDAC3 ( HD3 )-induced pAkt Ser-473 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. HUVECs were infected with Ad-null or Ad- XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by cellular fraction isolation and Western blot analysis. DMSO was included as vehicle control. The anti-FLAG antibody was included to detect exogenous XBP1u and HDAC3. Antibodies against α-tubulin and histone H3 were included to indicate cytosol and nuclear extract, respectively. The samples from cytosol and nuclear extraction were run on separate gels but performed Western blot at the same time and exposed to x-ray film exactly at the same time period. H , AZD2014 attenuated XBP1u/HDAC3-induced Akt1 phosphorylation in nucleus. HUVECs were infected with Ad-null or Ad- XBP1u or Ad-HDAC3 at 10 MOI for 24 h and then treated with 5 μmol/liter AZD2014 for 24 h, followed by double immunofluorescence staining with anti-mTOR ( red ) and anti-pAkt Ser-473 ( green ) antibodies. I , AZD2014 reduced flow-induced Nrf2 nuclear translocation. HUVECs were treated with 5 μmol/liter AZD2014 for 1 h, followed by disturbed flow for 4 h or being kept at static conditions in the presence of ZAD2014. DMSO was included as vehicle control. Double immunofluorescence staining was performed with anti-mTOR ( red ) and anti-Nrf2 ( green ) or pAkt Ser-473 ( green ) antibodies. Data presented are representatives of three independent experiments.

Article Snippet: Human Nrf2 siRNA (sc-37030) was purchased from Santa Cruz Biotechnology and reconstituted accordingly.

Techniques: Over Expression, Phospho-proteomics, Infection, Western Blot, Expressing, Control, Knockdown, shRNA, Transfection, Modification, Translocation Assay, Isolation, Extraction, Double Immunofluorescence Staining

Journal: The Journal of Biological Chemistry

Article Title: Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3 *

doi: 10.1074/jbc.M114.571984

Figure Lengend Snippet: XBP1 physically interacted with HDAC3. A , XBP1u and HDAC3 synergistically activated HO-1 expression. HUVECs were co-infected with Ad- XBP1u and Ad- HDAC3 at 10 MOI each for 24 h, followed by Western blot analysis. Ad-null virus was included as control and to compensate the MOI. FLAG antibody was used to detect exogenous XBP1u and HDAC3. B , knockdown of HDAC3 via shRNA lentivirus ( HDAC3sh ) attenuated Ad- XBP1u -induced HO-1 expression. Non-target shRNA lentivirus ( NTsh ) was included as control. C , XBP1u physically interacted with HDAC3. HEK293 cells were co-transfected with HA- XBP1u and FLAG- HDAC3 plasmids, followed by immunoprecipitation with anti-HA antibody and Western blot analysis with anti-FLAG and anti-HA antibodies. D , XBP1u bound to amino acid 201–323 region in HDAC3 molecule. The left panel indicates the schematic illustration of HDAC3 truncated mutants. The right panel shows the interaction of XBP1u and truncated HDAC3 as revealed by immunoprecipitation assays. E , disturbed flow increased XBP1u association with HDAC3/Akt1. Co-immunoprecipitation with anti-XBP1u antibody was performed on static and disturbed flow (4 h)-treated cells, followed by Western blot with anti-HDAC3 or Akt1 and anti-XBP1u antibodies. F , disturbed flow induced mTOR/Akt1/HDAC3/XBP1u complex formation in the cytoplasm. Double immunofluorescence staining was performed on static and disturbed flow (4 h)-treated cells. Antibodies are indicated with red or green letters reflecting the color in the images. Data presented are representatives of three independent experiments.

Article Snippet: Human Nrf2 siRNA (sc-37030) was purchased from Santa Cruz Biotechnology and reconstituted accordingly.

Techniques: Expressing, Infection, Western Blot, Virus, Control, Knockdown, shRNA, Transfection, Immunoprecipitation, Double Immunofluorescence Staining

Journal: The Journal of Biological Chemistry

Article Title: Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3 *

doi: 10.1074/jbc.M114.571984

Figure Lengend Snippet: A schematic illustration of flow-induced HO-1 expression. Disturbed flow ( D-Flow ) may activate VEGF receptor ( KDR ) in a ligand-independent manner, which in turn induces the complex formation among mTOR, Akt1, XBP1u, and HDAC3. The complex formation stabilizes both XBP1u and HDAC3 and activates Akt1 phosphorylation ( p ), leading to Nrf2 stabilization. Nrf2 translocates into nucleus and binds to the ARE in the HMOX-1 gene promoter and recruit co-activators ( ca ), promoting the HMOX-1 transcription. HO-1 catalyzes the heme degradation, which produces antioxidant biliverdin and carbon monoxide ( CO ), antagonizing disturbed flow-induced reactive oxygen species ( ROS ), leading to the maintenance of the redox homeostasis.

Article Snippet: Human Nrf2 siRNA (sc-37030) was purchased from Santa Cruz Biotechnology and reconstituted accordingly.

Techniques: Expressing, Phospho-proteomics