Journal: Nutrients

Article Title: The Food Contaminants Nivalenol and Deoxynivalenol Induce Inflammation in Intestinal Epithelial Cells by Regulating Reactive Oxygen Species Release

doi: 10.3390/nu9121343

Figure Lengend Snippet: ( A ) Effect of NIV, DON, and their combination (NIV + DON; 5 μM), on Nrf2 nuclear translocation, evaluated using immunofluorescence assay confocal microscopy. Scale bar: 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and Nrf2, respectively; ( B ) Effect of NIV; ( C ) DON; and ( D ) their combination (NIV + DON) on HO-1 expression in the IEC-6 cells, evaluated by cytofluorimetric technique. Values, mean ± s.e.m., are expressed as mean fluorescence intensity. ° Denotes p < 0.05 vs control. * Denotes p < 0.05 vs. LPS + IFN; ( E ) Flow cytometry figures show gated cells for HO-1; ( F – H ) Histograms representing the percentage of cells positive to anti-HO-1.

Article Snippet: After blocking with bovine serum albumin (BSA) and PBS, cells were incubated with rabbit anti-nitrotyrosine (Millipore, Billerica, MA, USA), rabbit anti-Nrf2 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA), or rabbit anti-phospho p65 NF-κB (Santa Cruz Biotechnologies, Dallas, TX, USA), for 1 h at 37 °C.

Techniques: Translocation Assay, Immunofluorescence, Confocal Microscopy, Expressing, Fluorescence, Control, Flow Cytometry

Journal: Pharmaceutical Biology

Article Title: Oxidative stress-mediated hepatotoxicity in rats induced by ethanol extracts of different parts of Chloranthus serratu s

doi: 10.1080/13880209.2020.1859552

Figure Lengend Snippet: Expression of heme oxygenase-1 (HO-1) and nuclear factor erythroid-2 related factor 2 (Nrf2) proteins in the liver tissues. The rats were treated with the ethanol extracts of different parts of C. serratus (ER: 4.14 g/kg/day, ES: 3.20 g/kg/day and EL: 1.16 g/kg/day) for 14 days. (A, C) Western blotting results of the Nrf2/HO-1 pathway. Graphs represented the relative expression of HO-1 (B) and Nrf2 (D). Compared with that in the Con group, the HO-1 level in the ER group was slightly decreased, whereas it was significantly decreased in the ES and EL groups. The Nrf2 levels in the drug-treated groups were significantly decreased. Data were presented as mean ± SD (n = 6). * p < 0.05, ** p < 0.01 vs. the Con group; # p < 0.05, ## p < 0.01 vs. the ES group.

Article Snippet: Rabbit anti-nuclear factor erythroid-2 related factor 2 (Nrf2) (cat: 16396-1-AP) and rabbit anti-β-tubulin (cat: 16385-1-AP) antibodies were obtained from Proteintech Co., Ltd. (Chicago, IL, USA).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Pro-Resolving Lipid Mediator Resolvin E1 Mitigates the Progress of Diethylnitrosamine-Induced Liver Fibrosis in Sprague-Dawley Rats by Attenuating Fibrogenesis and Restricting Proliferation

doi: 10.3390/ijms21228827

Figure Lengend Snippet: Effect of RvE1 on tissue levels of NF-κB and Nrf2, and molecules targets. Western blot analysis of ( A ) cytoplasmic and ( B ) nuclear NF-κB; and ( C ) cytoplasmic and ( D ) nuclear Nrf2. The cytoplasmic levels were normalized to GAPDH as housekeeping and nuclear levels were normalized to histone H1 as housekeeping. ( E , F ) serum levels of TNF-α and IL-10. ( G – I ) tissue levels of GSH, GSSG and GSH:GSSG ratio. n = 6–9 rats per experimental group. Asterisk indicates p < 0.05, and the letters identify the experiments that are compared and present this statistical difference.

Article Snippet: Mouse monoclonal primary antibodies used were for cyclin D1 (1:1000), BCL-2 (1:500), β-actin (1:2000), and GAPDH (1:2000), with these last two used as a cytoplasmic housekeeping protein (Nrf2, NF-κB, active caspase-3, histone H1, cyclin D1, GAPDH and secondary antibodies were purchased from Merck Millipore, Burlington, MA, USA; BCL-2 from Thermo Fischer Scientific, Rockford, IL, USA; and β-actin from Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot

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

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