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: Acta Pharmaceutica Sinica. B

Article Title: Nuclear TIGAR mediates an epigenetic and metabolic autoregulatory loop via NRF2 in cancer therapeutic resistance

doi: 10.1016/j.apsb.2021.10.015

Figure Lengend Snippet: Nuclear TIGAR acts as a novel NRF2 coactivator for activation of NSD2 expression and the antioxidant program. (A) Left panels: top, schematics of NSD2 promoter region with indicated locations of primers used in ChIP assay. Bottom, ChIP analysis of TIGAR occupancy on NSD2 promoter region in Tam-treated, MCF-7-TamR cells. ChIP data are presented as a percentage of input signals. Right panel, relative TIGAR occupancy at the NSD2 promoter (primer 4 site) in MCF-7 parental and TamR cells treated with vehicle or tamoxifen (TAM, 1 μmol/L). (B) ChIP analysis of NRF2 occupancy at NSD2 promoter region in MCF-7-TamR cells (left) and at the NSD2 promoter site 4 in the indicated cells treated with vehicle or Tam (right). Results shown are representative of at least three independent experiments. (C) ChIP analysis of relative occupancy by NRF2, TIGAR, MLL1, RNA polymerase II (pol II), RNA pol II serine 2 phosphorylation (pol II-pSer2), RNA pol II serine 5 phosphorylation (pol II-pSer5) and H3K4me3 and H3K27ac marks at the NSD2 promoter site (primer 4) in MCF-7-TamR cells transfected with control or TIGAR siRNA. (D) Reporter gene assay with NQO-1-ARE-luciferase (right) and NSD2-promoter-luciferase (left) was performed by transfecting 293T cells with vectors for the NQO1-ARE-dependent Firefly luciferase reporter and indicated constructs for expressing NRF2, TIGAR, TIGAR TM (triple mutation). β -Gal construct was included as an internal control. (E) qRT-PCR analysis of indicated gene expression in MCF-7 cells transfected with siTIGAR or control siRNA, and treated with vehicle or tBHQ (50 μmol/L). (F) qRT-PCR analysis of indicated gene expression in MCF-7-TamR cells transfected with NRF2 or control siRNA. (G) Whole cell lysates from MCF-7-TamR cells were prepared, and IP was performed with either anti-NRF2 antibody (top) or anti-TIGAR antibody (bottom) followed by IB with indicated antibodies. Results shown are representative of at least three independent experiments. The data are presented as the mean ± SD of triplicate of each sample ( n = 3; ∗ P < 0.05, ∗∗ P <0.01).

Article Snippet: Cells were the incubated with anti-TIGAR antibody (rabbit, Abcam; ab37910; mouse, Santa cruz; sc-166291), anti-NRF2 antibody (rabbit, Proteintech; 16396-1-AP; rabbit, GeneTex; GTX103322) or anti-V5-tag (mouse monoclonal, Abcam; ab27671), used at 1:500.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Transfection, Control, Reporter Gene Assay, Luciferase, Construct, Mutagenesis, Quantitative RT-PCR, Gene Expression

Journal: Acta Pharmaceutica Sinica. B

Article Title: Nuclear TIGAR mediates an epigenetic and metabolic autoregulatory loop via NRF2 in cancer therapeutic resistance

doi: 10.1016/j.apsb.2021.10.015

Figure Lengend Snippet: TIGAR physically interacts with NRF2 and promotes NRF2 nuclear translocation. (A) MCF-7 cells were transfected with TIGAR or control siRNA, after 3 days, cells treated with H 2 O 2 or vehicle for another 6 h, subcellular localization of TIGAR were examined by IF confocal microscopy. Cells were immunostained with NRF2 antibody (green). The nucleus is stained with DAPI (blue). Merged images (Merge) are shown. The scale bar represents 20 μm. (B) Subcellular localization of V5 tagged TIGAR and NRF2 were examined by IF confocal microscopy in A549 vector control cells or nuclear localization signal (NLS) linked TIGAR overexpressing cells. Cells were immunostained with V5 antibody (red) and NRF2 antibody (green). The nucleus was stained with DAPI (blue). Merged images (Merge) are shown. The scale bar represents 20 μm. (C) Whole cell lysates from MCF-7-TamR cells were prepared, and IP was performed with either anti-NRF2 antibody (top) or anti-TIGAR antibody (bottom) followed by IB with indicated antibodies. (D) Top left, schematics of His-tagged human NRF2 protein with numbers indicating boundary amino acid number at the indicated domains. GST pulldown assays were performed with GST-wild (WT) or mutant (TM) TIGAR and full length His-tagged NRF2 or its deletion forms. His-p97/VCP was used as a negative control.

Article Snippet: Cells were the incubated with anti-TIGAR antibody (rabbit, Abcam; ab37910; mouse, Santa cruz; sc-166291), anti-NRF2 antibody (rabbit, Proteintech; 16396-1-AP; rabbit, GeneTex; GTX103322) or anti-V5-tag (mouse monoclonal, Abcam; ab27671), used at 1:500.

Techniques: Translocation Assay, Transfection, Control, Confocal Microscopy, Staining, Plasmid Preparation, Mutagenesis, Negative Control