Journal: Experimental and Therapeutic Medicine

Article Title: Sinomenine activation of Nrf2 signaling prevents inflammation and cerebral injury in a mouse model of ischemic stroke

doi: 10.3892/etm.2021.10079

Figure Lengend Snippet: SINO treatment relieves MCAO-induced cerebral injuries. (A) Representative micrographs of H&E staining of brain sections (x20 magnification; scale bar, 50 µm). (B) Brain water content analysis in brain tissues from Sham (sham-operated, n=6), SINO (treatment only, n=6), MCAO (MCAO-operated, n=6) and SINO/MCAO (MCAO operated plus SINO treatment, n=6). (C) Western blot analysis of n-Nrf2, HO-1 and NQO1 protein expression levels from brain tissue. The samples from two randomly selected brains in each group were presented. (D) Quantification of brain protein expression levels. All the experiments were repeated at least three times. The data are presented as the mean ± SEM. * P<0.05 and ** P<0.01 vs. Sham; # P<0.05 and ## P<0.01 vs. MCAO. HO-1, heme oxygenase-1; MCAO, middle cerebral artery occlusion; n-Nrf2, nuclear-nuclear factor-erythroid 2-related factor; NQO1, NAD(P)H: Quinoneoxidoreductase 1; SINO, sinomenine.

Article Snippet: The following antibodies were used in the present study: SINO (MedChemExpress); ML385 (MedChemExpress); Nrf2, Keap1, NAD(P)H: Quinoneoxidoreductase (NQO1), Lamin B and HO-1 antibodies (Abcam); β-actin antibodies (Bioworld Technology, Inc.); NF-κB p65, Phosphorylated (p-)IκBα and IκBα antibodies (Cell Signaling Technology, Inc.); HRP-conjugated secondary antibodies (Bioworld Technology, Inc.); and Dylight488 conjugated goat anti-rabbit IgG secondary antibodies (Bioworld Technology, Inc.).

Techniques: Staining, Western Blot, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Sinomenine activation of Nrf2 signaling prevents inflammation and cerebral injury in a mouse model of ischemic stroke

doi: 10.3892/etm.2021.10079

Figure Lengend Snippet: SINO treatment activates the Nrf2 signaling pathway. (A) BV2 cells were treated with a serial of doses of SINO for 24 h. HO-1 and NQO1 protein expression levels were assayed using western blotting. (B) BV2 cells were treated with SINO (200 µM) for various durations. HO-1 and NQO1 protein expression levels were assayed using western blotting. (C) BV2 cells were treated with SINO (200 µM) for 4 h. n-Nrf2 and t-Nrf2 protein expression levels were assayed using western blotting. Nuclear protein Lamin B and β-actin were used as controls. (D) BV2 cells were treated with serial doses of SINO for 24 h. The mRNA expression levels of HO-1 and NQO1 were measured using reverse transcription-semi-quantitative PCR. (E) Cell viability assay. BV2 cells were treated with various doses of SINO for 24 h. The cell viability was assayed by a CCK-8 kit. All the experiments were repeated at least three times. * P<0.05 and ** P<0.01 vs. Control HO-1, heme oxygenase-1; NQO1, NAD(P)H: Quinoneoxidoreductase 1; n-, nuclear; Nrf2; nuclear factor-erythroid 2-related factor; SINO, sinomenine; t-, total.

Article Snippet: The following antibodies were used in the present study: SINO (MedChemExpress); ML385 (MedChemExpress); Nrf2, Keap1, NAD(P)H: Quinoneoxidoreductase (NQO1), Lamin B and HO-1 antibodies (Abcam); β-actin antibodies (Bioworld Technology, Inc.); NF-κB p65, Phosphorylated (p-)IκBα and IκBα antibodies (Cell Signaling Technology, Inc.); HRP-conjugated secondary antibodies (Bioworld Technology, Inc.); and Dylight488 conjugated goat anti-rabbit IgG secondary antibodies (Bioworld Technology, Inc.).

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Viability Assay, CCK-8 Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Sinomenine activation of Nrf2 signaling prevents inflammation and cerebral injury in a mouse model of ischemic stroke

doi: 10.3892/etm.2021.10079

Figure Lengend Snippet: SINO treatment regulates microglia polarization and inflammation in an Nrf2-dependent manner. BV2 cells were stimulated with OGD for 4 h followed by treatment with SINO (200 µM) for 12 h. The mRNA expression levels of (A) M1 markers (IL-6/NOS2) and (B) M2 markers (IL-10/Arg-1) were determined using RT-sqPCR. BV2 cells were pretreated with ML385 (5 µM) for 48 h to inhibit Nrf2 expression. Cells were then stimulated with OGD for 4 h followed by treatment with SINO (200 µM) for 12 h. The mRNA expression levels of (C) M1 and (D) M2 markers were measured using RT-sqPCR. The data are presented as the mean ± SEM of three independent experiments. * P<0.05 and ** P<0.01 vs. Control; ## P<0.01 vs. OGD. Arg-1, arginase-1; NOS2, nitric oxide synthase 2; OGD, oxygen and glucose deprivation; SINO, sinomenine; RT-sqPCR, reverse transcription-semi-quantitative PCR.

Article Snippet: The following antibodies were used in the present study: SINO (MedChemExpress); ML385 (MedChemExpress); Nrf2, Keap1, NAD(P)H: Quinoneoxidoreductase (NQO1), Lamin B and HO-1 antibodies (Abcam); β-actin antibodies (Bioworld Technology, Inc.); NF-κB p65, Phosphorylated (p-)IκBα and IκBα antibodies (Cell Signaling Technology, Inc.); HRP-conjugated secondary antibodies (Bioworld Technology, Inc.); and Dylight488 conjugated goat anti-rabbit IgG secondary antibodies (Bioworld Technology, Inc.).

Techniques: Expressing, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Sinomenine activation of Nrf2 signaling prevents inflammation and cerebral injury in a mouse model of ischemic stroke

doi: 10.3892/etm.2021.10079

Figure Lengend Snippet: SINO treatment regulates microglia inflammation in an Nrf2-dependent manner. (A) BV2 cells were pretreated with ML385 (5 µM) for 48 h to inhibit Nrf2 expression. Cells were then stimulated with OGD for 4 h followed by treatment with SINO (200 µM) for 12 h. The protein expression levels of p-IκBα and total IκBα were analyzed using western blotting. (B) Immuno-fluorescent staining of cells was performed using an NF-κB p65 primary antibody and Dylight488 conjugated secondary antibody (middle row of panels). The cells were also stained with DAPI (top row of panels) and merged with NF-κB images (lower row of panels). The data are presented as the mean ± SEM of three independent experiments. ** P<0.01 vs. Control; ## P<0.01 vs. OGD. OGD, oxygen and glucose deprivation; p-, phosphorylated; SINO, sinomenine.

Article Snippet: The following antibodies were used in the present study: SINO (MedChemExpress); ML385 (MedChemExpress); Nrf2, Keap1, NAD(P)H: Quinoneoxidoreductase (NQO1), Lamin B and HO-1 antibodies (Abcam); β-actin antibodies (Bioworld Technology, Inc.); NF-κB p65, Phosphorylated (p-)IκBα and IκBα antibodies (Cell Signaling Technology, Inc.); HRP-conjugated secondary antibodies (Bioworld Technology, Inc.); and Dylight488 conjugated goat anti-rabbit IgG secondary antibodies (Bioworld Technology, Inc.).

Techniques: Expressing, Western Blot, Staining, Control

Journal: Food Science & Nutrition

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

doi: 10.1002/fsn3.70975

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

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

Techniques: Western Blot, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 deficiency enhances GSH recovery through Nrf2 signaling. ( A, B ) Bcl3 fl/fl and Bcl3 hep-/- mice were treated with APAP (300 mg/kg, intraperitoneally), and liver tissues were collected at 0, 3, 6, and 24 hours post-treatment. ( A ) Western blot analysis of Cyp2E1 levels in liver tissues. ( B ) Quantification of GSH levels in liver tissues using a GSH assay kit (n = 3–5 per group). ( C ) Immunohistochemical staining of GCLC in the livers of Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg, intraperitoneally) for 0 and 6 hours. ( D ) Western blot analysis of Nrf2, GCLC, and GCLM protein levels in liver tissues. ( E ) Quantification of protein levels. ( F ) qPCR analysis of Nrf2 , Gclc, and Gclm mRNA levels in liver tissues (n = 4–6 per group). Data represent 3 independent experiments and are presented as mean ± SEM. Statistical significance was determined by Student’s t-test ( B, E, F ); ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Western Blot, GSH Assay, Immunohistochemical staining, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 deficiency mitigates APAP-induced primary hepatocyte death. ( A ) Primary hepatocytes were isolated from Bcl3 hep-/- mice and Bcl3 fl/fl mice. Representative images of primary hepatocytes treated with APAP (10 mM) for 0 and 6 hours. ( B ) Cell viability of primary hepatocytes measured using the CCK8 assay. ( C ) GSH levels in primary hepatocytes after APAP treatment for 3 and 6 hours. ( D ) MitoSOX staining in primary hepatocytes detected by immunofluorescence (scale bar, 10 μm). ( E ) Quantification of MitoSOX mean fluorescence intensity in primary hepatocytes by flow cytometry. ( F ) Protein levels of Cyp2E1, Nrf2, GCLC, and GCLM in primary hepatocytes treated with APAP for 3 and 6 hours, as determined by Western blotting. Data represent 3 independent experiments. Data are presented as mean ± SEM. Student’s t -test ( B, C ), ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Isolation, CCK-8 Assay, Staining, Immunofluorescence, Fluorescence, Flow Cytometry, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 interacts with Nrf2. ( A ) Liver tissues from Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg intraperitoneally) for 0 and 6 hours were immunostained with anti-Bcl3 ( green ) and anti-Nrf2 ( red ) antibodies imaged using a confocal microscope (scale bar, 50 μm). ( B ) Primary hepatocytes isolated from Bcl3 hep-/- and Bcl3 fl/fl mice were immunostained for anti-Bcl3 ( green ) and anti-Nrf2 ( red ) following APAP treatment for 0 and 6 hours (scale bar, 10 μm). ( C–D ) Immunoprecipitation analysis of the interaction between Flag-Bcl3 and endogenous Nrf2 as well as Nrf2 and endogenous Bcl3 in Huh7 cells ( C ) and HEK293T cells ( D ) respectively. ( E ) Co-immunoprecipitation analysis using an anti-Bcl3 antibody to pull down Nrf2 protein in liver tissues from WT mice, followed by Western blotting.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Microscopy, Isolation, Immunoprecipitation, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Bcl3 knockout induces Nrf2 nuclear translocation following APAP treatment. ( A ) Liver tissues from Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg intraperitoneally) for 6 hours were immunostained for Nrf2 ( red ) (scale bar, 10 μm). ( B ) Primary hepatocytes isolated from Bcl3 hep-/- mice and Bcl3 fl/fl mice were immunostained for Nrf2 ( red ) following APAP treatment for 6 hours (scale bar, 10 μm). ( C ) Liver tissues from Bcl3 fl/fl and Bcl3 hep-/- mice treated with APAP (300 mg/kg intraperitoneally) for 6 hours were subjected to cytoplasmic and nuclear protein extraction. Nrf2 levels in cytoplasmic and nuclear fractions were analyzed by Western blotting (n = 3 per group). ( D ) Quantification of Western blot results. ( E ) Primary hepatocytes were isolated from the Bcl3 hep-/- mice and Bcl3 fl/fl mice. Nrf2 levels in cytoplasmic and nuclear fractions were analyzed by Western blotting (n = 3 per group). ( F ) Quantification of Western blot results. Data represent 3 independent experiments and are presented as mean ± SEM. Student t -test ( D, F ), ∗ P < .05; ∗∗ P < .01.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Knock-Out, Translocation Assay, Isolation, Protein Extraction, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: An Nrf2 inhibitor counteract the protective effect of Bcl3 on APAP-induced liver injury. ( A ) Experimental scheme. Bcl3 fl/fl and Bcl3 hep-/- mice were treated with the Nrf2 inhibitor ML385 (30 mg/kg/day intraperitoneally) for 5 days, fasted for 16 hours, and then administered APAP (300 mg/kg, intraperitoneally) for 24 hours before sacrifice and tissue collection. ( B ) Representative liver morphologies of mice from ( A ). ( C ) H&E staining of liver sections from mice in ( A ) (scale bar, 50 μm). ( D ) Quantification of necrotic areas in liver tissues (n = 5 per group). ( E ) Serum ALT levels (n = 5 per group). Data represent 3 independent experiments and are presented as mean ± SEM. Student t -test ( D, E ), ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Antibodies Used for Western Blot and Immunofluorescence

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver-specific Bcl3 Knockout Alleviates Acetaminophen-induced Liver Injury by Activating Nrf2 Pathway in Male Mice

doi: 10.1016/j.jcmgh.2025.101483

Figure Lengend Snippet: Primers Used for qRT-PCR Analysis

Article Snippet: Liver sections or MPHs were incubated with primary antibodies against Nrf2 (Proteintech), GCLC (Proteintech), and Bcl3 (Santa Cruz) at 4 °C overnight.

Techniques: Sequencing

Journal: Antioxidants

Article Title: Selective Inhibition of Vascular Smooth Muscle Cell Function by COVID-19 Antiviral Drugs: Impact of Heme Oxygenase-1

doi: 10.3390/antiox14080945

Figure Lengend Snippet: RDV stimulates HO-1 promoter activity via the Nrf2/ARE complex in rat aortic SMCs. ( A ) RDV-mediated HO-1 mRNA expression is dependent on de novo RNA synthesis. SMCs were treated with actinomycin D (ActD; 1 µg/mL) for 8 h in the absence or presence of RDV (50 µM). ( B ) RDV stimulates HO-1 promoter activity. Cells were transfected with a HO-1 promoter construct (E1) or a mutated HO-1 construct (M739) and a Renilla luciferase construct, treated with RDV (50 µM) for 8 h, and then analyzed for luciferase activity. In some instances, a dominant-negative Nrf2 (dnNrf2) construct was co-transfected into cells. ( C ) RDV (50 µM), but not MPR (50 µM) or NTV (50 µM), exposure for 24 h increases ROS production in SMCs. ( D ) RDV (50 µM) exposure for 24 h stimulates ROS production that is blocked by the antioxidant N-acetyl-L-cysteine (NAC; 10 mM). ( E ) ChIP assays demonstrate that RDV (50 µM) exposure for 8 h increases Nrf2 binding to the HO-1 enhancer E1 that is blocked by NAC (10 mM). ( F ) NAC (10 mM) inhibits RDV (50 µM for 24 h)-mediated HO-1 protein expression. Statistical analysis was performed using an analysis of variance with the Holm–Sidak post hoc test. * Statistically significant effect of RDV.

Article Snippet: Polyclonal antibodies against HO-1 and HO-2 were from Enzo Life Sciences (Farmingdale, NY, USA) while antibodies directed against NF-E2-related factor-2 (Nrf2) and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Expressing, Transfection, Construct, Luciferase, Dominant Negative Mutation, Binding Assay

Journal: Antioxidants & Redox Signaling

Article Title: Inhibition of NFE2L2-Antioxidant Response Element Pathway by Mitochondrial Reactive Oxygen Species Contributes to Development of Cardiomyopathy and Left Ventricular Dysfunction in Chagas Disease

doi: 10.1089/ars.2016.6831

Figure Lengend Snippet: NFE2L2 transcriptional activation in chagasic myocardium (±MnSOD). Mice were infected with T. cruzi and harvested at 150 days pi. (A) Western blotting. Heart tissue homogenates (A.a, A.c) and nuclear fractions (A.b, A.d) were subjected to Western blotting with anti-NFE2L2 antibody (A.a, A.b), and densitometry analysis of the NFE2L2 signal (A.c, A.d) is presented. (B) Western blotting and densitometry analyses of Keap1 (B.a, B.b) and HO1 (B.a, B.c) levels in heart homogenates. Densitometry analysis of WB bands in heart homogenates and nuclear fractions were normalized to GAPDH and Lamin A/C, respectively. (C) NFE2L2–ARE binding. NFE2L2 double-stranded (ds) DNA-binding capacity in nuclear fractions of heart tissue from WT and MnSODtg mice (±Tc) was measured as described in Materials and Methods section. (D) RT-qPCR analysis for γGCS mRNA level in WT and MnSODtg mice (±Tc). Data are plotted as mean value ± SEM (n = 6–10 mice per group). Significance is shown as ***,###p < 0.001. ARE, antioxidant response element; γGCS, gamma-glutamyl cysteine synthase; HO1, heme oxygenase-1; Keap1, Kelch-like ECH-associated protein 1; mRNA, messenger RNA; NFE2L2, nuclear factor (erythroid 2)-like 2; RT-qPCR, real-time quantitative polymerase chain reaction.

Article Snippet: Membranes were blocked with 5% nonfat dry milk (NFDM) in 50 m M Tris-HCl (pH 7.5)/150 m M NaCl (TBS), washed with TBS–0.1% Tween 20 (TBST) and TBS, and incubated overnight at 4°C with antirabbit antibodies against NFE2L2 (Cat. No. 12721S, 1:1000; Cell Signaling), Keap1 (Cat. No. sc33569, 1:1000; Santa Cruz, Inc., Santa Cruz, CA), HO1 (Cat. No. 5061, 1:1000; Cell Signaling), MnSOD (Cat. No. PA1-31072, 1:5000; Life Technology, Carlsbad, CA), COX IV (Cat. No. ab16056, 1:2000; Abcam, San Francisco, CA), Lamin A/C (Cat. No. sc20681, 1:1000; Santa Cruz, Inc.), and GAPDH (Cat. No. 3683, 1:1000; Cell Signaling).

Techniques: Activation Assay, Infection, Western Blot, Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Antioxidants & Redox Signaling

Article Title: Inhibition of NFE2L2-Antioxidant Response Element Pathway by Mitochondrial Reactive Oxygen Species Contributes to Development of Cardiomyopathy and Left Ventricular Dysfunction in Chagas Disease

doi: 10.1089/ars.2016.6831

Figure Lengend Snippet: MnSOD preserves NFE2L2–ARE functional activity in Tc-infected cells. (A) Confocal microscopy. Cardiac myocytes were transfected with empty vector (A.a–A.f) or pBI.EGFP–MnSOD (A.g–A.l) and incubated with (A.d–A.f, A.j–A.l) or without (A.a–A.c, A.g–A.i) T. cruzi for 24 h. Cells were stained with DAPI (nuclear blue, A.a, A.d, A.g, A.j) and Alexa Fluor 568-conjugated anti-NFE2L2 antibody (red, A.b, A.e, A.h, A.k) and analyzed by confocal fluorescence microscopy. Overlay images (A.c, A.f, A.i, A.l) show cytosolic and nuclear localization of NFE2L2. (B) Antioxidant genes' expression. RT-qPCR measurement of mRNA levels for (B.a) NFE2L2, (B.b) GCLM, (B.c) Trx, (B.d) GST, and (B.e) NQO1 in cardiomyocytes transfected with MnSOD expression plasmid and infected with T. cruzi for 24 h. Fold change was determined after normalizing the data with GAPDH mRNA. (C) NFE2L2 transcriptional activity. HeLa cells were transiently transfected with pGL3-promo-2x-hHO1 (C.a), pGL3-promo-2x-hNQO1 (C.b), or pGL3-promo-2x-hGCLM (C.c) plasmids consisting of luciferase encoding gene under NFE2L2-binding ARE sequence from HO1, NQO1, and GCLM, respectively. Cells were cotransfected with an MnSOD-expressing plasmid and a Renilla luciferase plasmid (positive control for normalization of transfection efficiency). Transfected cells were infected (cell:Tcratio, 1:5) for 24 h. The relative NFE2L2 transcriptional activity for HO1, NQO1, and GCLM was measured by using a dual luciferase assay and normalized to Renilla luciferase activity. Data (mean ± SEM) are representative of three independent experiments (triplicate observations per experiment). Significance is shown as *p < 0.05, **,##p < 0.01, ***,###p < 0.001. GCLM, glutamate-cysteine ligase modifier subunit; GST, glutathione S transferase; NQO1, NAD(P)H dehydrogenase, quinone 1; Trx, thioredoxin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: Membranes were blocked with 5% nonfat dry milk (NFDM) in 50 m M Tris-HCl (pH 7.5)/150 m M NaCl (TBS), washed with TBS–0.1% Tween 20 (TBST) and TBS, and incubated overnight at 4°C with antirabbit antibodies against NFE2L2 (Cat. No. 12721S, 1:1000; Cell Signaling), Keap1 (Cat. No. sc33569, 1:1000; Santa Cruz, Inc., Santa Cruz, CA), HO1 (Cat. No. 5061, 1:1000; Cell Signaling), MnSOD (Cat. No. PA1-31072, 1:5000; Life Technology, Carlsbad, CA), COX IV (Cat. No. ab16056, 1:2000; Abcam, San Francisco, CA), Lamin A/C (Cat. No. sc20681, 1:1000; Santa Cruz, Inc.), and GAPDH (Cat. No. 3683, 1:1000; Cell Signaling).

Techniques: Functional Assay, Activity Assay, Infection, Confocal Microscopy, Transfection, Plasmid Preparation, Incubation, Staining, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Luciferase, Binding Assay, Sequencing, Positive Control

Journal: Respiratory Research

Article Title: Intermittent hypoxia promotes melanoma lung metastasis via oxidative stress and inflammation responses in a mouse model of obstructive sleep apnea

doi: 10.1186/s12931-018-0727-x

Figure Lengend Snippet: Effect of OSA-like IH on oxidative stress responses in melanoma lung metastasis mice. qRT-PCR analysis of SOD ( A ) and p22 phox ( B ) mRNA expression ( n = 6 per group) and western blotting analysis of NRF2 protein expression ( C a ) in tumor tissue from mouse lungs after OSA-like IH exposure and/or tempol treatment ( n = 3 per group). The expression level of NRF2 was evaluated by densitometric analysis of western blot bands ( C b ). All data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: The primary antibodies, including HIF-1α (1:200; Novus Biologicals, Littleton, CO, USA), NF-E2-related factor 2 (NRF2) (1:200; ProteinTech Group, Chicago, IL, USA), NF-κB P65 (1:5000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and β-actin (1:5000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), were used to probe the target proteins.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Lack of the immune adaptor molecule SARM1 accelerates disease in prion infected mice and is associated with increased mitochondrial respiration and decreased expression of NRF2

doi: 10.1371/journal.pone.0267720

Figure Lengend Snippet: (A) Representative immunoblots for MFN2, Drp1, OPA1, NRF2 and mouse β-actin. Data are from C57Bl/6 (left two panels) or SARM1 KO mice (right two panels) inoculated IC with normal brain homogenate (NBH) or RML prions (RML). The brackets on the far left indicate the two bands representing L-OPA1 (L) and the two bands representing S-OPA1 (S). The antibodies used are described in the materials and methods. Quantitation of (B) MFN2, (C) Drp-1, (D) OPA1 long (L-OPA1) and short (S-OPA1), and (E) NRF2 in C57Bl/6 and SARM1 KO mice inoculated with NBH or RML prions. Data were normalized to mouse β-actin (Relative intensity) and were calculated from n = 5 animals for each condition. For panels B-E, mean ± SEM is shown. Significance was calculated using the unpaired Student’s t-test with Welch’s correction. * p = 0.02; ** p = 0.003–0.004; *** p = 0.0003–0.0006. Black bars = NBH inoculated, open bars = RML prion inoculated.

Article Snippet: The protein NRF2 was detected using an anti-NRF2 mouse monoclonal antibody (Novus Biologicals, Littleton CO) at a dilution of 1:500.

Techniques: Western Blot, Quantitation Assay

Journal: Biomedical reports

Article Title: Mistimed H 2 S upregulation, Nrf2 activation and antioxidant proteins levels in renal tubular epithelial cells subjected to anoxia and reoxygenation.

doi: 10.3892/br.2020.1309

Figure Lengend Snippet: Figure 4. Expression of Nrf2 and its transcriptional targets under anoxia or reoxygenation. (A) Expression of Nrf2 and its transcriptional targets (B) SOD‑3, (C) glutathione reductase, (D) ferritin H and (E) xCT increased under anoxia, and returned to baseline levels during reoxygenation. Representative blots are presented. *P<0.001 vs. Ctrl; #P<0.001 vs. Reox. Nrf2, nuclear factor erythroid 2‑like 2; Ctrl, control; Reox, reoxygenation; OD, optical density; SOD‑3, super‑ oxide dismutase 3; xCT, cystine‑glutamate antiporter.

Article Snippet: Primary antibodies used were specific for CBS (1:1,000; cat. no. TA338394; OriGene Technologies Inc.), CSE (1:100; cat. no. sc‐374249; Santa Cruz Biotechnology, Inc.), 3‐MST (1:100; cat. no. sc‐376168; Santa Cruz Biotechnology, Inc.), Nrf2 (1:1,000; cat. no. TA343586; OriGene Technologies, Inc.), BIOMEDICAL REPORTS 13: 3, 2020 3 superoxide dismutase 3 (SOD3; 1:100; cat. no. sc‐271170; Santa Cruz Biotechnology, Inc.), glutathione reductase (GR; 1:100; cat. no. sc‐133245; Santa Cruz Biotechnology, Inc.), ferritin heavy chain (1:100; cat. no. sc‐376594; Santa Cruz Biotechnology, Inc.), cystine‐glutamate antiporter (xCT; 1:1,000; cat. no. ANT‐111; Alomone Labs), activated cleaved‐caspase‐3 (175) (1:500; cat. no. 7074; Cell Signaling Technology, Inc.), p53 (1:500; cat. no. 2524 Cell Signaling Technology, Inc.), p53 phosphorylated at serine 15 (p‐p53) (1:500; cat. no. 9284; Cell Signaling Technology, Inc.), Bax (1:500; cat. no. 5023; Cell Signaling Technology, Inc.) and β‐actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Expressing, Control