c57bl 6j nrf2  (Jackson Laboratory)

Journal: iScience

Article Title: Hypomorphic biliverdin reductase a mutations define bilirubin anti-malarial threshold

doi: 10.1016/j.isci.2026.115958

Figure Lengend Snippet: Expression analysis of BVRA missense mutants G17A and E97A in C57BL/6J mice (A) RT-qPCR analysis of Blvra mRNA expression in spleen, kidney, and liver from Blvra WT , Blvra −/− and Blvra G17A mice. Data are expressed as Blvra / Rplp0 (2 −ΔCt ) and presented as individual data points with bar graphs showing mean ± SD. n = 3 mice per genotype. (B) Western blot analysis of BVRA protein expression in spleen, kidney, and liver. Representative blots show BVRA (33 kDa) and β-actin (42 kDa) loading control. Quantification of BVRA protein levels normalized to β-actin is shown below each blot as individual data points with bar graphs showing mean ± SD. n = 3–5 per genotype. (C) RT-qPCR analysis of Blvra mRNA expression in spleen, kidney and liver tissues from Blvra WT , Blvra −/− , and Blvra E97A mice. Data are expressed as Blvra / Rplp0 (2 −ΔCt ) and presented as individual data points with bar graphs showing mean ± SD. n = 3 per genotype. (D) Western blot analysis of BVRA protein expression in spleen, kidney, and liver tissue. Representative blots and quantification of BVRA protein levels normalized to β-actin below each blot, shown as individual data points with bar graphs showing mean ± SD. n = 3–6 mice per genotype. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; NS, not significant.

Article Snippet: C57BL/6J Nrf2 −/− , Jackson Laboratory , Cat#017009; RRID: IMSR_JAX:017009.

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

Journal: iScience

Article Title: Hypomorphic biliverdin reductase a mutations define bilirubin anti-malarial threshold

doi: 10.1016/j.isci.2026.115958

Figure Lengend Snippet: BVRA oxidoreductase activity is dispensable for heme-induced NRF2 activation and insulin receptor signaling in BMDM (A) BMDM from Blvra WT , Blvra −/− , Blvra G17A , and Blvra E97A mice were stimulated with heme and NRF2 activation assessed by immunofluorescence. (B) Representative immunofluorescence images of NRF2 nuclear translocation in BMDM following heme stimulation. Images show confocal z stack cross-sections with orthogonal views. NRF2 is shown in red, DAPI nuclear stain in blue, and phalloidin (cytoskeleton marker) in white. Scale bars, 5 μm. (C) Quantification of cytoplasmic (left graph) or nuclear (right graph) NRF2 MFI in BMDM stimulated with (+) or without (−) heme. Each data point represents a technical replicate. n = 5–10 replicates. Data in (C) is representative from two independent experiments with a similar trend. (D) BMDM from Blvra WT , Blvra −/− , Blvra G17A , and Blvra E97A mice were stimulated with heme and NRF2-regulated gene expression assessed by qRT-PCR. (E) RT-qPCR analysis of NRF2 target genes ( Nqo1 , Fth , Gclc , Hmox1 , Nrf2 , and Blvra ) in BMDM stimulated with (+) or without (−) heme. Data expressed as gene/ Rplp0 (2 −ΔCt ). Each data point represents a technical replicate. n = 3–6 replicates from two independent experiments with a similar trend. (F) BMDM from Blvra WT , Blvra −/− , Blvra G17A , and Blvra E97A mice were stimulated with insulin and INSR signaling assessed by western blot. (G) Representative western blots showing IRS-1 phosphorylation at tyrosine 612 (IRS-1 Y612 ), at serine 307 (IRS-1 S307 ), total IRS-1, and vinculin as loading control in BMDM stimulated with insulin vs. control vehicle. (H) Quantification of IRS-1 Y612 normalized to IRS-1 S307 in BMDM stimulated with (+) or without (−) insulin. Each data point represents a technical replicate. n = 3 replicates from one experiment. Data in (C, E, and H) are presented as individual data points with bar graphs showing mean ± SD. Statistical comparisons were performed using two-way ANOVA with Šídák’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01; NS, not significant.

Article Snippet: C57BL/6J Nrf2 −/− , Jackson Laboratory , Cat#017009; RRID: IMSR_JAX:017009.

Techniques: Activity Assay, Activation Assay, Immunofluorescence, Translocation Assay, Staining, Marker, Gene Expression, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Control

Journal: Redox Biology

Article Title: Nrf2 mitigates prolonged PM2.5 exposure-triggered liver inflammation by positively regulating SIKE activity: Protection by Juglanin

doi: 10.1016/j.redox.2020.101645

Figure Lengend Snippet: PM 2.5 incubation inhibits Nrf2 and SIKE activation in vitro . (A) RT-qPCR and western blot results of Nrf2, and (B) RT-qPCR analysis of HO-1, NQO-1, GCLC and GCLM in L02 cells incubated with 24 h of PM 2.5 (0–100 μg/ml as indicated). (C) RT-qPCR and western blot results of Nrf2, and (D) RT-qPCR results of HO-1, NQO-1, GCLC and GCLM in L02 cells exposed to PM 2.5 (100 μg/ml) for the shown time. (E) RT-qPCR and western blot analysis of SIKE, and (F) western blot results for p-TBK1 and p-NF-κB/p65 in L02 cells treated with different concentrations of PM 2.5 as indicated for 24 h. (G) RT-qPCR and western blot analysis of SIKE, and (H) western blot results for p-TBK1 and p-NF-κB in L02 cells cultured with PM 2.5 (100 μg/ml) for the indicated time. Data are expressed as means ± SEM (n = 3 independent observations). *P < 0.05 and **P < 0.01 versus the Con group without any treatments.

Article Snippet: 2) Nrf2 knockout C57BL/6J mice (Nrf2 −/− ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Incubation, Activation Assay, In Vitro, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: Redox Biology

Article Title: Nrf2 mitigates prolonged PM2.5 exposure-triggered liver inflammation by positively regulating SIKE activity: Protection by Juglanin

doi: 10.1016/j.redox.2020.101645

Figure Lengend Snippet: Nrf2 positively regulates SIKE expression in PM 2.5 -incubated cells. (A) L02 cells were transfected with Nrf2 specific siRNA for 24 h, and then were collected for transfection efficiency determination using western blotting analysis. (B) RT-qPCR analysis of HO-1, NQO-1, GCLC and GCLM in L02 cells transfected with siNrf2 for 24 h. (C) Western blot (left panel) and RT-qPCR (right panel) analysis of SIKE in L02 cells with siNrf2 transfection for 24 h. (D) Luciferase reporter analysis with HEK-293 cells that were co-transfected with the indicated reporter plasmids plus siNC or siNrf2 and then left untreated or treated with PM 2.5 (0, 25, 50 or 100 μg/ml) for 24 h. (E) L02 cells were transfected with empty plasmid (EP) of Nrf2 plasmids (Nrf2) for 24 h. Cells were then collected for western blotting analysis to measure Nrf2 expression levels. (F) RT-qPCR analysis of HO-1, NQO-1, GCLC and GCLM in L02 cells transfected with Nrf2 plasmids for 24 h. (G) Western blot (left panel) and RT-qPCR (right panel) analysis of SIKE in L02 cells transfected with 24 h of Nrf2 plasmids. (H) Luciferase reporter analysis with HEK-293 cells transfected with the indicated reporter plasmids plus EP or Nrf2 plasmids and then left untreated or treated with increasing concentrations of PM 2.5 for 24 h. (I) L02 cells were treated with PM 2.5 for 24 h, followed by immunofluorescence staining of Nrf2 (green fluorescence) and SIKE (red fluorescence). Scale bar was 25 μm. (J) Binding of Nrf2 to SIKE by ChIP assay. L02 cells were treated with or without PM 2.5 (100 μg/ml) for 24 h. Cross-linked chromatin was immunoprecipitated with an antibody to Nrf2, in the absence of antibody (input), or an isotype-matched control (IgG). Isolated DNA was purified and analyzed by PCR. (K) L02 cells were transfected with siNrf2 for 24 h, and were then incubated with 100 μg/ml of PM 2.5 for another 24 h. Subsequently, all cells were collected for western blot analysis of SIKE, p-TBK1 and p-NF-κB. (L) L02 cells were transfected with Nrf2 plasmids for 24 h, followed by PM 2.5 (100 μg/ml) incubation for another 24 h. Then, western blot analysis was used to determine SIKE, p-TBK1 and p-NF-κB protein levels. Data are expressed as means ± SEM (n = 3 independent observations). *P < 0.05 and **P < 0.01; ns , no significant difference.

Article Snippet: 2) Nrf2 knockout C57BL/6J mice (Nrf2 −/− ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Expressing, Incubation, Transfection, Western Blot, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence, Binding Assay, Immunoprecipitation, Control, Isolation, Purification

Journal: Redox Biology

Article Title: Nrf2 mitigates prolonged PM2.5 exposure-triggered liver inflammation by positively regulating SIKE activity: Protection by Juglanin

doi: 10.1016/j.redox.2020.101645

Figure Lengend Snippet: Juglanin improves Nrf2 activation to inhibit oxidative stress in PM 2.5 -incubated cells. (A) L02 cells were treated with different concentrations of Jug (0–80 μM) for 24 h. Then, morphology of cells was observed. (B) Left, L02 cells were incubated with Jug at the indicated concentrations for 24 h, and were then collected for cell viability calculation using western blotting analysis. Right, L02 cells were treated with 40 μM of Jug for the shown time, followed by MTT analysis to assess the cell viability. (C) Western blotting results for nuclear and cytoplasm Nrf2 expression levels in L02 cells incubated with increasing concentrations of Jug for 24 h. (D) Immunofluorescence staining of Nrf2 in Jug-treated L02 cells for 24 h. Scale bar was 25 μm. (E–G) L02 cells were treated with PM 2.5 (100 μg/ml) for 24 h together with Jug (40 μM) or t-BHQ (10 μM). Then, all cells were collected for further analysis as follows. (E) Western blot analysis of Keap-1 and HO-1. (F) Assessments of SOD, CAT and GPX activities in cells. (G) DCF-DA staining of L02 cells. Data are expressed as means ± SEM. *P < 0.05 and **P < 0.01; ns , no significant difference.

Article Snippet: 2) Nrf2 knockout C57BL/6J mice (Nrf2 −/− ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Nrf2 mitigates prolonged PM2.5 exposure-triggered liver inflammation by positively regulating SIKE activity: Protection by Juglanin

doi: 10.1016/j.redox.2020.101645

Figure Lengend Snippet: Effects of Nrf2 activation on PM 2.5 -induced hepatic injury in mice. (A) Body weight of mice was recorded (n = 8 per group). (B) MBP results for mice from each group (n = 8 per group). (C) Liver weight of mice (n = 8 per group). (D) H&E staining of hepatic sections (n = 5 per group). Scale bar was 50 μm. (E) SOD and MDA measurements in liver samples (n = 8 per group). (F) RT-qPCR analysis of HO-1, NQO-1, GCLC and GCLM in liver tissues. (G) Immunofluorescence staining of SIKE in hepatic sections (n = 5 per group). Scale bar was 50 μm. (H) Quantification of SIKE fluorescence intensity. (I) Western blot results for SIKE in liver samples (n = 5 per group). (J) RT-qPCR results for hepatic IL-1β, IL-6, TNF-α and IFN-β mRNA expression levels (n = 5 per group). (K) Western blot analysis of liver p-TBK1 and p-NF-κB (n = 5 per group). Data are expressed as means ± SEM. *P < 0.05 and **P < 0.01; ns , no significant difference.

Article Snippet: 2) Nrf2 knockout C57BL/6J mice (Nrf2 −/− ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

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

Journal: Redox Biology

Article Title: Nrf2 mitigates prolonged PM2.5 exposure-triggered liver inflammation by positively regulating SIKE activity: Protection by Juglanin

doi: 10.1016/j.redox.2020.101645

Figure Lengend Snippet: Juglanin ameliorates PM 2.5 -induced hepatic injury through improving Nrf2/SIKE signaling pathway in mice. (A) Body weight change of mice (n = 8 per group). (B) MBP results for mice (n = 8 per group). (C) Liver weight of mice (n = 8 per group). (D) Up panel, H&E staining for liver samples; down panel, immunofluorescence staining of Nrf2 (green) and SIKE (red) in liver sections (n = 5 per group). Scale bar was 50 μm. (E) Western blot analysis for liver Nrf2, SIKE, p-TBK1 and p-NF-κB (n = 5 per group). (F) RT-qPCR results for hepatic Nrf2 mRNA expression levels (n = 5 per group). (G) SOD and MDA in liver tissues were measured (n = 8 per group). (H) RT-qPCR results for hepatic IL-1β, IL-6, TNF-α, IFN-β, HO-1, NQO-1, GCLC and GCLM (n = 5 per group). Data are expressed as means ± SEM. *P < 0.05 and **P < 0.01; ns , no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 2) Nrf2 knockout C57BL/6J mice (Nrf2 −/− ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

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

Journal: Redox Biology

Article Title: Nrf2 mitigates prolonged PM2.5 exposure-triggered liver inflammation by positively regulating SIKE activity: Protection by Juglanin

doi: 10.1016/j.redox.2020.101645

Figure Lengend Snippet: Proposed mechanism of Nrf2/SIKE-mediated hepatic injury induced by long-term PM 2.5 exposure. After PM 2.5 long-term challenge, down-regulated activation of Nrf2 repressed SIKE expression, contributing to oxidative stress and inflammatory response through reducing anti-oxidants and facilitating TBK1/NF-κB signaling. These effects led to hepatic injury consequently. However, Juglanin treatment showed protective effects against PM 2.5 -induced liver injury by improving Nrf2/SIKE signaling pathway.

Article Snippet: 2) Nrf2 knockout C57BL/6J mice (Nrf2 −/− ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Activation Assay, Expressing

Journal: Free radical biology & medicine

Article Title: Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

doi: 10.1016/j.freeradbiomed.2019.12.012

Figure Lengend Snippet: RUT prevented DSS-induced acute colitis depending on NRF2. (A and B), body weight change for Nrf2+/+ mice (A) and Nrf2−/− mice (B). (C and D), DAI score for Nrf2+/+ mice (C) and Nrf2−/− mice (D). (E and F), colon length of Nrf2+/+ mice (E) and Nrf2−/− mice (F). *P < 0.05, **P < 0.01*** and P < 0.001, versus vehicle-treated DSS group. Significance was determined by using one-way ANOVA. Data are presented as the mean ± SEM, N = 6 per group.

Article Snippet: Male 6- to 8-week-old C57BL/6J wild-type ( Nrf2 +/+ ) and Nrf2 -null ( Nrf2 −/− ) mice on the C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques:

Journal: Free radical biology & medicine

Article Title: Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

doi: 10.1016/j.freeradbiomed.2019.12.012

Figure Lengend Snippet: H&E staining analyses of distal colon and ileum sections. (A), H&E staining analyses of colon sections for both Nrf2+/+ and Nrf2−/− mice. (B), H& E staining analyses of ileum sections for both Nrf2+/+ and Nrf2−/− mice. Scale bar size = 100 μm.

Article Snippet: Male 6- to 8-week-old C57BL/6J wild-type ( Nrf2 +/+ ) and Nrf2 -null ( Nrf2 −/− ) mice on the C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Staining

Journal: Free radical biology & medicine

Article Title: Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

doi: 10.1016/j.freeradbiomed.2019.12.012

Figure Lengend Snippet: Analysis of proinflammatory gene mRNAs in colon and ileum. (A), Expression of proinflammatory gene mRNAs in colons of Vehicle-, DSS-and DSS+RUT-treated Nrf2+/+ mice; (B), Expression of proinflammatory gene mRNAs in ileum of Vehicle-, DSS- and DSS+RUT-treated Nrf2+/+ mice. (C), Expression of proinflammatory gene mRNAs in colon of Vehicle-, DSS-and DSS+RUT-treated Nrf2−/− mice. (D), Expression of proinflammatory gene mRNAs in ileum of Vehicle-, DSS- and DSS+RUT-treated Nrf2−/− mice. *P < 0.05, **P < 0.01, and ***P < 0.001, versus DSS group. Statistical significance was determined by one-way ANOVA. Data are presented as the mean ± SEM, N = 6 for each group.

Article Snippet: Male 6- to 8-week-old C57BL/6J wild-type ( Nrf2 +/+ ) and Nrf2 -null ( Nrf2 −/− ) mice on the C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Expressing

Journal: Free radical biology & medicine

Article Title: Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

doi: 10.1016/j.freeradbiomed.2019.12.012

Figure Lengend Snippet: Analysis of NRF2 target gene mRNAs in colon and ileum or primary intestinal epithelial cells. (A and B), Expression of NRF2 target gene mRNAs in colon (A) and ileum (B) from Nrf2+/+ and Nrf2−/− mice treated with DSS +/− RUT. (C), Expression of NRF2 target gene mRNAs in RUT or SFN-treated primary intestinal epithelial cells. *P < 0.05 and **P < 0.01 versus the Nrf2+/+ DSS-treated group for Panels A and B. *P < 0.05, *P < 0.01 and ***P < 0.001 versus the Vehicle-treated group for Panel C, Data are presented as the mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test or one-way ANOVA. N = 6 for each group.

Article Snippet: Male 6- to 8-week-old C57BL/6J wild-type ( Nrf2 +/+ ) and Nrf2 -null ( Nrf2 −/− ) mice on the C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Expressing, Two Tailed Test

Journal: Free radical biology & medicine

Article Title: Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

doi: 10.1016/j.freeradbiomed.2019.12.012

Figure Lengend Snippet: Immunofluorescent assay for NRF2 nuclear translocation after RUT treatment. (A), Double immunofluorescent staining for nucleus (Hoechst, blue color) and NRF2 protein (FITC, green color) by control vehicle or 10 μM of RUT in HCT116 cells, magnification time, 400×. N = 3 for each group. (B), Representative western blot analysis of cytosol NRF2 and nuclear NRF2 levels in RUT or SFN-treated HCT116 cells. NRF2, when phosphorylated to be activated, was detected at 116 kDa. β-Actin (ACTB) and Lamin B (LMNB1) were used a cytosolic and nuclear loading controls. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Male 6- to 8-week-old C57BL/6J wild-type ( Nrf2 +/+ ) and Nrf2 -null ( Nrf2 −/− ) mice on the C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Translocation Assay, Staining, Control, Western Blot

Journal: Free radical biology & medicine

Article Title: Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

doi: 10.1016/j.freeradbiomed.2019.12.012

Figure Lengend Snippet: RUT activates NRF2 by interfering with the interaction between KEAP1 and NRF2. (A), SPR assay for interaction of RUT with kelch domain KEAP1 protein; (B), SPR assay for NRF2-KEAP1 interaction treated with 0, 12.5, 37.5 μM of RUT; (C) Ligand docking of RUT in the kelch domain of human KEAP1 protein; (D and E) Luciferase assays for NRF2 activation in HCT116 (D) and HepG2 (E). Data are presented as the mean ± SEM, n = 3 per group. **P < 0.01, ***P < 0.001, versus control group, was determined by one-way ANOVA test.

Article Snippet: Male 6- to 8-week-old C57BL/6J wild-type ( Nrf2 +/+ ) and Nrf2 -null ( Nrf2 −/− ) mice on the C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: SPR Assay, Luciferase, Activation Assay, Control

Journal: Free radical biology & medicine

Article Title: Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis

doi: 10.1016/j.freeradbiomed.2019.12.012

Figure Lengend Snippet: Proposed mechanism and pathway of RUT in suppressing DSS-induced IBD in mice. In cytoplasm, KEAP1 protein is usually bound to NRF2 to stabilize NRF2 in the cytoplasm. Upon oxidative stress treatment, RUT directly binds to KEAP1 resulting in NRF2 release and translocation to the nucleus to bind to AREs and initiate the anti-oxidative response by upregulating the expression of downstream target genes including Nqo1, Hmox1, and Sod1, which then decreases intracellular ROS production and alleviates oxidative stress-induced cytotoxicity. Pharmacologically, RUT improved DSS-induced colitis and alleviated intestinal inflammation dependent on NRF2 activation.

Article Snippet: Male 6- to 8-week-old C57BL/6J wild-type ( Nrf2 +/+ ) and Nrf2 -null ( Nrf2 −/− ) mice on the C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Translocation Assay, Expressing, Activation Assay