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Digoxin decreased Nrf2 at transcriptional level through inhibiting PI3K/Akt pathway in SW1990/Gem and Panc-1/Gem cells. (A–B) Effects of digoxin on protein levels of p-JNK, JNK, p-ERK1/2, ERK1/2, p-P38 and P38. (C–D) Effects of digoxin on protein levels of PI3K, p-Akt, Akt. (E–F) SW1990/Gem and Panc-1/Gem cells were treated with 80 nM of digoxin, 20 µM of LY294002, or a combination of digoxin and LY294002 for 24 h, the protein levels of Nrf2, NQO1, <t>HO-1,</t> and GCLC were detected by Western blot. (G–H) SW1990/Gem and Panc-1/Gem cells were treated with 80 nM of digoxin, 20 µM of LY294002, or a combination of digoxin and LY294002 for 24 h, Nrf2 mRNA levels were detected by qRT-PCR. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P
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1) Product Images from "Digoxin sensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine via inhibiting Nrf2 signaling pathway"

Article Title: Digoxin sensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine via inhibiting Nrf2 signaling pathway

Journal: Redox Biology

doi: 10.1016/j.redox.2019.101131

Digoxin decreased Nrf2 at transcriptional level through inhibiting PI3K/Akt pathway in SW1990/Gem and Panc-1/Gem cells. (A–B) Effects of digoxin on protein levels of p-JNK, JNK, p-ERK1/2, ERK1/2, p-P38 and P38. (C–D) Effects of digoxin on protein levels of PI3K, p-Akt, Akt. (E–F) SW1990/Gem and Panc-1/Gem cells were treated with 80 nM of digoxin, 20 µM of LY294002, or a combination of digoxin and LY294002 for 24 h, the protein levels of Nrf2, NQO1, HO-1, and GCLC were detected by Western blot. (G–H) SW1990/Gem and Panc-1/Gem cells were treated with 80 nM of digoxin, 20 µM of LY294002, or a combination of digoxin and LY294002 for 24 h, Nrf2 mRNA levels were detected by qRT-PCR. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P
Figure Legend Snippet: Digoxin decreased Nrf2 at transcriptional level through inhibiting PI3K/Akt pathway in SW1990/Gem and Panc-1/Gem cells. (A–B) Effects of digoxin on protein levels of p-JNK, JNK, p-ERK1/2, ERK1/2, p-P38 and P38. (C–D) Effects of digoxin on protein levels of PI3K, p-Akt, Akt. (E–F) SW1990/Gem and Panc-1/Gem cells were treated with 80 nM of digoxin, 20 µM of LY294002, or a combination of digoxin and LY294002 for 24 h, the protein levels of Nrf2, NQO1, HO-1, and GCLC were detected by Western blot. (G–H) SW1990/Gem and Panc-1/Gem cells were treated with 80 nM of digoxin, 20 µM of LY294002, or a combination of digoxin and LY294002 for 24 h, Nrf2 mRNA levels were detected by qRT-PCR. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P

Techniques Used: Western Blot, Quantitative RT-PCR

Digoxin increased the sensitivity of SW1990/Gem and Panc-1/Gem cells to gemcitabine by inhibiting Nrf2 signaling. (A–B) Effects of Nrf2 knockdown on protein levels of Nrf2, NQO1, HO-1, and GCLC in SW1990/Gem and Panc-1/Gem cells. (C–D) Effects of Nrf2 knockdown on reversing drug resistance of gemcitabine in SW1990/Gem and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P
Figure Legend Snippet: Digoxin increased the sensitivity of SW1990/Gem and Panc-1/Gem cells to gemcitabine by inhibiting Nrf2 signaling. (A–B) Effects of Nrf2 knockdown on protein levels of Nrf2, NQO1, HO-1, and GCLC in SW1990/Gem and Panc-1/Gem cells. (C–D) Effects of Nrf2 knockdown on reversing drug resistance of gemcitabine in SW1990/Gem and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P

Techniques Used:

Nrf2 signaling was upregulated in SW1990/Gem and Panc-1/Gem cells. (A–B) The cytotoxicity of gemcitabine to SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. (C–D) Western blot was used to detect the protein level of Nrf2, NQO1, HO-1, and GCLC in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. (E–F) The expression levels of Nrf2 target genes in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. The colors of the heatmap reflect log 2 -expression levels of Nrf2 target genes in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P
Figure Legend Snippet: Nrf2 signaling was upregulated in SW1990/Gem and Panc-1/Gem cells. (A–B) The cytotoxicity of gemcitabine to SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. (C–D) Western blot was used to detect the protein level of Nrf2, NQO1, HO-1, and GCLC in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. (E–F) The expression levels of Nrf2 target genes in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. The colors of the heatmap reflect log 2 -expression levels of Nrf2 target genes in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P

Techniques Used: Western Blot, Expressing

Digoxin inhibited the expressions of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. (A–B) Effects of digoxin on the expressions of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. The colors of the heatmap reflect log 2 -expression levels of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. (C–D) Effects of digoxin on protein levels of NQO1, HO-1 and GCLC in SW1990/Gem and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as **P
Figure Legend Snippet: Digoxin inhibited the expressions of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. (A–B) Effects of digoxin on the expressions of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. The colors of the heatmap reflect log 2 -expression levels of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. (C–D) Effects of digoxin on protein levels of NQO1, HO-1 and GCLC in SW1990/Gem and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as **P

Techniques Used: Expressing

Digoxin sensitized SW1990/Gem cells-derived xenografts to gemcitabine treatment by inhibiting Nrf2 signaling. (A–D) Digoxin sensitized SW1990/Gem-shControl cells-derived xenografts to gemcitabine treatment. (E–H) Digoxin could not sensitize SW1990/Gem-shNrf2 cells-derived xenografts to gemcitabine treatment. (I) Effects of digoxin on the protein levels of Nrf2, NQO1, HO-1, and GCLC in tumor tissues. (J) Tumor tissues were subjected to IHC-Nrf2, IHC-NQO1, IHC-HO-1, IHC-GCLC, IHC-Ki67 and TUNEL staining. All images were shown at ×200. Data were expressed as mean ± SD, n = 6. Significant differences were indicated as ***P
Figure Legend Snippet: Digoxin sensitized SW1990/Gem cells-derived xenografts to gemcitabine treatment by inhibiting Nrf2 signaling. (A–D) Digoxin sensitized SW1990/Gem-shControl cells-derived xenografts to gemcitabine treatment. (E–H) Digoxin could not sensitize SW1990/Gem-shNrf2 cells-derived xenografts to gemcitabine treatment. (I) Effects of digoxin on the protein levels of Nrf2, NQO1, HO-1, and GCLC in tumor tissues. (J) Tumor tissues were subjected to IHC-Nrf2, IHC-NQO1, IHC-HO-1, IHC-GCLC, IHC-Ki67 and TUNEL staining. All images were shown at ×200. Data were expressed as mean ± SD, n = 6. Significant differences were indicated as ***P

Techniques Used: Derivative Assay, Immunohistochemistry, TUNEL Assay, Staining

2) Product Images from "Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer"

Article Title: Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer

Journal: PLoS Medicine

doi: 10.1371/journal.pmed.0030420

Increased NRF2 Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean ± SD for each concentration.
Figure Legend Snippet: Increased NRF2 Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean ± SD for each concentration.

Techniques Used: Activity Assay, MTT Assay, Concentration Assay

Mutant KEAP1 Protein Is Unable to Suppress NRF2 Activity (A) Repression activity of the KEAP1 mutants was monitored by a luciferase reporter assay. Wild-type and mutant KEAP1 cDNA constructs were transfected onto H838 cells stably expressing ARE luciferase reporter. Data represent mean ± SD ( n = 3). (B) Silencing of NRF2 by siRNA in A549 cells downregulated the expression of NRF2-dependent genes. A nonspecific siRNA (NS siRNA) was used as control. (C) Inhibition of KEAP1 expression by siRNA in BEAS2B cells upregulated the expression of NRF2-dependent genes.
Figure Legend Snippet: Mutant KEAP1 Protein Is Unable to Suppress NRF2 Activity (A) Repression activity of the KEAP1 mutants was monitored by a luciferase reporter assay. Wild-type and mutant KEAP1 cDNA constructs were transfected onto H838 cells stably expressing ARE luciferase reporter. Data represent mean ± SD ( n = 3). (B) Silencing of NRF2 by siRNA in A549 cells downregulated the expression of NRF2-dependent genes. A nonspecific siRNA (NS siRNA) was used as control. (C) Inhibition of KEAP1 expression by siRNA in BEAS2B cells upregulated the expression of NRF2-dependent genes.

Techniques Used: Mutagenesis, Activity Assay, Luciferase, Reporter Assay, Construct, Transfection, Stable Transfection, Expressing, Inhibition

Status of KEAP1 and NRF2 Is Altered in Cancer Cells (A) Immunoblot showing increased nuclear localization of NRF2 in nuclear extracts (NE) from cancer cells. Cancer cells showed lower levels of KEAP1 (~69 kDa) and higher levels of NRF2 (~110 kDa) in total protein lysates (TP). NIVT and KIVT indicate NRF2 and KEAP1 in vitro transcribed/translated product, respectively. (B and C) Quantification of NRF2 and KEAP1 protein in immunoblots. For band densitometry, bands in nuclear extract blot (B) were normalized to Lamin B1, and those in total protein (C) were normalized to GAPDH. (D) Heat map showing relative expression of KEAP1, NRF2, and NRF2-dependent genes by real-time RT-PCR. Raw data for the heat maps are presented in Table S5 .
Figure Legend Snippet: Status of KEAP1 and NRF2 Is Altered in Cancer Cells (A) Immunoblot showing increased nuclear localization of NRF2 in nuclear extracts (NE) from cancer cells. Cancer cells showed lower levels of KEAP1 (~69 kDa) and higher levels of NRF2 (~110 kDa) in total protein lysates (TP). NIVT and KIVT indicate NRF2 and KEAP1 in vitro transcribed/translated product, respectively. (B and C) Quantification of NRF2 and KEAP1 protein in immunoblots. For band densitometry, bands in nuclear extract blot (B) were normalized to Lamin B1, and those in total protein (C) were normalized to GAPDH. (D) Heat map showing relative expression of KEAP1, NRF2, and NRF2-dependent genes by real-time RT-PCR. Raw data for the heat maps are presented in Table S5 .

Techniques Used: In Vitro, Western Blot, Expressing, Quantitative RT-PCR

Dysfunctional KEAP1–NRF2 Interaction in NSCLC Tumors (A) Immunohistochemical analysis of NRF2 in NSCLC tissues. Part a shows a patient (PT-18) with mutation in KEAP1 showing strong nuclear and cytoplasmic staining. Part b shows a patient negative for mutation (PT-28) showing weak cytoplasmic staining. Part c shows a patient negative for mutation (PT-20) showing increased nuclear and cytoplasmic staining in tumor tissue. Part d shows weakly staining normal bronchus from the same patient (PT-20). (B) Total GSH and enzyme activities of NQO1 and total GST in NSCLC and matched normal tissues. Raw data for the heat maps are presented in Table S4 . *, samples harboring KEAP1 mutation; §, nmol/mg protein; †, nmol DCPIP reduced/min/mg protein; ‡, nmol of product formed/min/mg protein.
Figure Legend Snippet: Dysfunctional KEAP1–NRF2 Interaction in NSCLC Tumors (A) Immunohistochemical analysis of NRF2 in NSCLC tissues. Part a shows a patient (PT-18) with mutation in KEAP1 showing strong nuclear and cytoplasmic staining. Part b shows a patient negative for mutation (PT-28) showing weak cytoplasmic staining. Part c shows a patient negative for mutation (PT-20) showing increased nuclear and cytoplasmic staining in tumor tissue. Part d shows weakly staining normal bronchus from the same patient (PT-20). (B) Total GSH and enzyme activities of NQO1 and total GST in NSCLC and matched normal tissues. Raw data for the heat maps are presented in Table S4 . *, samples harboring KEAP1 mutation; §, nmol/mg protein; †, nmol DCPIP reduced/min/mg protein; ‡, nmol of product formed/min/mg protein.

Techniques Used: Immunohistochemistry, Mutagenesis, Staining

3) Product Images from "Testosterone propionate activated the Nrf2-ARE pathway in ageing rats and ameliorated the age-related changes in liver"

Article Title: Testosterone propionate activated the Nrf2-ARE pathway in ageing rats and ameliorated the age-related changes in liver

Journal: Scientific Reports

doi: 10.1038/s41598-019-55148-0

Immunohistochemistry revealed variations in Nrf2, HO-1 and NQO1 immunoreactive intensity in the liver tissue of aged rats. Bar = 200 μm. Bar graph shows the AOD of Nrf2 ( a ), HO-1 ( b ) and NQO1 ( c ). The asterisks show significant differences (* P
Figure Legend Snippet: Immunohistochemistry revealed variations in Nrf2, HO-1 and NQO1 immunoreactive intensity in the liver tissue of aged rats. Bar = 200 μm. Bar graph shows the AOD of Nrf2 ( a ), HO-1 ( b ) and NQO1 ( c ). The asterisks show significant differences (* P

Techniques Used: Immunohistochemistry

The effect of TP on STAT5b, Keap1 and the Nrf2-ARE signalling pathway mRNA in the liver tissue of aged rats. Bar graphs show STAT5b mRNA ( a ), Keap1 mRNA ( b ), Nrf2 mRNA ( c ), HO-1 mRNA ( d ) and NQO1 mRNA ( e ). The asterisks show significant differences (* P
Figure Legend Snippet: The effect of TP on STAT5b, Keap1 and the Nrf2-ARE signalling pathway mRNA in the liver tissue of aged rats. Bar graphs show STAT5b mRNA ( a ), Keap1 mRNA ( b ), Nrf2 mRNA ( c ), HO-1 mRNA ( d ) and NQO1 mRNA ( e ). The asterisks show significant differences (* P

Techniques Used:

The effect of TP on STAT5b, Keap1 and the Nrf2-ARE signalling pathway proteins in the liver tissue of aged rats. Western blot analysis revealed STAT5b, Keap1, Nrf2, HO-1 and NQO1 proteins expression in liver tissue. ( a ) Bar graphs illustrate the protein expression of STAT5b ( a ), Keap1 ( b ), Nrf2 ( c ), HO-1 ( d ) and NQO1 ( e ). The asterisks show significant differences (* P
Figure Legend Snippet: The effect of TP on STAT5b, Keap1 and the Nrf2-ARE signalling pathway proteins in the liver tissue of aged rats. Western blot analysis revealed STAT5b, Keap1, Nrf2, HO-1 and NQO1 proteins expression in liver tissue. ( a ) Bar graphs illustrate the protein expression of STAT5b ( a ), Keap1 ( b ), Nrf2 ( c ), HO-1 ( d ) and NQO1 ( e ). The asterisks show significant differences (* P

Techniques Used: Western Blot, Expressing

4) Product Images from "Sustained Activation of Nuclear Erythroid 2-Related Factor 2/Antioxidant Response Element Signaling Promotes Reductive Stress in the Human Mutant Protein Aggregation Cardiomyopathy in Mice"

Article Title: Sustained Activation of Nuclear Erythroid 2-Related Factor 2/Antioxidant Response Element Signaling Promotes Reductive Stress in the Human Mutant Protein Aggregation Cardiomyopathy in Mice

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2010.3587

Sustained Nrf2 nuclear translocation in the TG hearts induces gene expression of ARE-dependent antioxidants. Real-time RT-PCR determinations of Nrf2 target genes in NTG and TG mouse at 3 (A–H) and 6 months (I–P) were performed using Qiagen-mouse
Figure Legend Snippet: Sustained Nrf2 nuclear translocation in the TG hearts induces gene expression of ARE-dependent antioxidants. Real-time RT-PCR determinations of Nrf2 target genes in NTG and TG mouse at 3 (A–H) and 6 months (I–P) were performed using Qiagen-mouse

Techniques Used: Translocation Assay, Expressing, Quantitative RT-PCR

Schematic proposal for Nrf2 activation and mechanism for reductive stress in the mutant-protein aggregation cardiomyopathy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article
Figure Legend Snippet: Schematic proposal for Nrf2 activation and mechanism for reductive stress in the mutant-protein aggregation cardiomyopathy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article

Techniques Used: Activation Assay, Mutagenesis

Decreased ubiquitination and increased activity of Nrf2 in the TG mouse. (A) TransAM-Nrf2 activity assay: Nuclear extracts from NTG and TG mouse ( n = 6) at 6 months were incubated with the precoated antioxidant response element (ARE) oligonucleotides.
Figure Legend Snippet: Decreased ubiquitination and increased activity of Nrf2 in the TG mouse. (A) TransAM-Nrf2 activity assay: Nuclear extracts from NTG and TG mouse ( n = 6) at 6 months were incubated with the precoated antioxidant response element (ARE) oligonucleotides.

Techniques Used: Activity Assay, Incubation

Sequestration of Keap1 in to the mutant CryAB aggregates facilitate Nrf2 nuclear translocation. Immunofluorescence analysis was performed using the CryAB (Rb.ab, green) and Keap1 (SC-Goat ab, red) and merged with the nuclear staining (DRAQ1). CryAB and
Figure Legend Snippet: Sequestration of Keap1 in to the mutant CryAB aggregates facilitate Nrf2 nuclear translocation. Immunofluorescence analysis was performed using the CryAB (Rb.ab, green) and Keap1 (SC-Goat ab, red) and merged with the nuclear staining (DRAQ1). CryAB and

Techniques Used: Mutagenesis, Translocation Assay, Immunofluorescence, Staining

5) Product Images from "Beclin-1 regulates cigarette smoke–induced kidney injury in a murine model of chronic obstructive pulmonary disease"

Article Title: Beclin-1 regulates cigarette smoke–induced kidney injury in a murine model of chronic obstructive pulmonary disease

Journal: JCI Insight

doi: 10.1172/jci.insight.99592

Autophagy activity is induced in mouse kidneys after cigarette smoke exposure. ( A ) Scheme representing autophagic flux experiment in which leupeptin or bafilomycin A1 inhibits autophagosome degradation, leading to autophagosome accumulation. ( B ) Mice subjected to RA or CS for 2 months (left panel) or 6 months (right panel) were assayed for autophagic flux in vivo by injection with leupeptin or vehicle (PBS), followed by Western blotting for LC3B expression in kidney tissue after 2 and 6 months of exposure and Beclin-1 expression after 6 months of exposure. Dot plots represent quantitation of Western blots ( n = 3 per group, except for 6 months CS + leupeptin exposure, n = 2 per group). ( C ) HK-2 cells were exposed to CSE in the absence or presence of bafilomycin A1. Representative Western blot of Beclin-1 and LC3B expression to determine autophagic flux in vitro. Dot plots represent quantitation of 3 independent experiments. All data are mean ± SEM.* P
Figure Legend Snippet: Autophagy activity is induced in mouse kidneys after cigarette smoke exposure. ( A ) Scheme representing autophagic flux experiment in which leupeptin or bafilomycin A1 inhibits autophagosome degradation, leading to autophagosome accumulation. ( B ) Mice subjected to RA or CS for 2 months (left panel) or 6 months (right panel) were assayed for autophagic flux in vivo by injection with leupeptin or vehicle (PBS), followed by Western blotting for LC3B expression in kidney tissue after 2 and 6 months of exposure and Beclin-1 expression after 6 months of exposure. Dot plots represent quantitation of Western blots ( n = 3 per group, except for 6 months CS + leupeptin exposure, n = 2 per group). ( C ) HK-2 cells were exposed to CSE in the absence or presence of bafilomycin A1. Representative Western blot of Beclin-1 and LC3B expression to determine autophagic flux in vitro. Dot plots represent quantitation of 3 independent experiments. All data are mean ± SEM.* P

Techniques Used: Activity Assay, Mouse Assay, In Vivo, Injection, Western Blot, Expressing, Quantitation Assay, In Vitro

6) Product Images from "Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling"

Article Title: Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling

Journal: The FASEB Journal

doi: 10.1096/fj.201601155R

Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD.  A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis.  B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin.  C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Translocation Assay, CTL Assay

Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells.  A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA.  D ,  E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls.  F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Translocation Assay, CTL Assay

7) Product Images from "PALB2 Interacts with KEAP1 To Promote NRF2 Nuclear Accumulation and Function"

Article Title: PALB2 Interacts with KEAP1 To Promote NRF2 Nuclear Accumulation and Function

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06271-11

Competition between PALB2 and NRF2 for KEAP1 binding. (A) Disruption of preformed NRF2/KEAP1 complex in cell extracts. Increasing amounts of whole-cell extracts from 293T cells transfected with pOZC1-PALB2WT or pOZC1-PALB2T92E (lanes 2 and 6, respectively) were added to equal aliquots of extract made from cells cotransfected with HA-NRF2 and Myc-KEAP-GFP. Then, KEAP1 was precipitated using a GFP antibody and the amounts of proteins were analyzed by Western blotting. (B and C) Competitive disruption of isolated NRF2/KEAP1 or PALB2/KEAP1 complexes by IVTed PALB2T551 or NRF2, respectively. See Materials and Methods for detailed procedures. (D) In vitro competition between PALB2 and NRF2 for KEAP1 binding. Different amounts of PALB2 and NRF2 were generated together with a fixed amount of KEAP1 in triple IVT reactions by using different combinations of amounts (in μl) of PCR-generated templates for KEAP1, PALB2, and NRF2 (top panels). Then, KEAP1 was IPed and the amounts of KEAP1, PALB2, and NRF2 in the precipitates were analyzed by Western blotting using anti-HA antibody, which recognizes all 3 proteins (bottom panels).
Figure Legend Snippet: Competition between PALB2 and NRF2 for KEAP1 binding. (A) Disruption of preformed NRF2/KEAP1 complex in cell extracts. Increasing amounts of whole-cell extracts from 293T cells transfected with pOZC1-PALB2WT or pOZC1-PALB2T92E (lanes 2 and 6, respectively) were added to equal aliquots of extract made from cells cotransfected with HA-NRF2 and Myc-KEAP-GFP. Then, KEAP1 was precipitated using a GFP antibody and the amounts of proteins were analyzed by Western blotting. (B and C) Competitive disruption of isolated NRF2/KEAP1 or PALB2/KEAP1 complexes by IVTed PALB2T551 or NRF2, respectively. See Materials and Methods for detailed procedures. (D) In vitro competition between PALB2 and NRF2 for KEAP1 binding. Different amounts of PALB2 and NRF2 were generated together with a fixed amount of KEAP1 in triple IVT reactions by using different combinations of amounts (in μl) of PCR-generated templates for KEAP1, PALB2, and NRF2 (top panels). Then, KEAP1 was IPed and the amounts of KEAP1, PALB2, and NRF2 in the precipitates were analyzed by Western blotting using anti-HA antibody, which recognizes all 3 proteins (bottom panels).

Techniques Used: Binding Assay, Transfection, Western Blot, Isolation, In Vitro, Generated, Polymerase Chain Reaction

PALB2 binds KEAP1 through a highly conserved ETGE motif. (A) Schematic of PALB2 constructs used in the domain mapping study. (B and C) PALB2 constructs were transiently expressed in 293T cells, proteins were IPed with anti-FLAG M2 agarose, and the precipitates were analyzed by Western blotting. (D) Amino acid sequence alignment of the N-terminal sequences of PALB2 and NRF2 showing the shared LDEETGE KEAP1 binding motif. (E) Deletion or mutations of the ETGE motif in PALB2 all abolish KEAP1 binding. (F) E91R and T92E mutations do not affect the binding of PALB2 to BRCA2, RAD51, and BRCA1.
Figure Legend Snippet: PALB2 binds KEAP1 through a highly conserved ETGE motif. (A) Schematic of PALB2 constructs used in the domain mapping study. (B and C) PALB2 constructs were transiently expressed in 293T cells, proteins were IPed with anti-FLAG M2 agarose, and the precipitates were analyzed by Western blotting. (D) Amino acid sequence alignment of the N-terminal sequences of PALB2 and NRF2 showing the shared LDEETGE KEAP1 binding motif. (E) Deletion or mutations of the ETGE motif in PALB2 all abolish KEAP1 binding. (F) E91R and T92E mutations do not affect the binding of PALB2 to BRCA2, RAD51, and BRCA1.

Techniques Used: Construct, Western Blot, Sequencing, Binding Assay

PALB2 interacts with KEAP1. (A) Domain structure and known binding partners of PALB2. (B) Tandem affinity purification of PALB2 from a HeLa S3 cell line stably expressing a FLAG-HA-double-tagged PALB2 protein (PALB2-FH). Numbers at left are molecular masses in kilodaltons. (C) Reciprocal co-IP of endogenous PALB2 and KEAP1 in U2OS cells. (D) Endogenous KEAP1 association with PALB2 and NRF2 under normal and stress conditions. U2OS cells were treated with DMSO (mock), H 2 O 2 , tBHQ, and CPT at the indicated concentrations for 2 h, and the interactions were analyzed by IP-Western blotting.
Figure Legend Snippet: PALB2 interacts with KEAP1. (A) Domain structure and known binding partners of PALB2. (B) Tandem affinity purification of PALB2 from a HeLa S3 cell line stably expressing a FLAG-HA-double-tagged PALB2 protein (PALB2-FH). Numbers at left are molecular masses in kilodaltons. (C) Reciprocal co-IP of endogenous PALB2 and KEAP1 in U2OS cells. (D) Endogenous KEAP1 association with PALB2 and NRF2 under normal and stress conditions. U2OS cells were treated with DMSO (mock), H 2 O 2 , tBHQ, and CPT at the indicated concentrations for 2 h, and the interactions were analyzed by IP-Western blotting.

Techniques Used: Binding Assay, Affinity Purification, Stable Transfection, Expressing, Co-Immunoprecipitation Assay, Cycling Probe Technology, Western Blot

A proposed model of PALB2 regulation of NRF2 in the nucleus. Nuclear NRF2 forms heterodimers with small Maf proteins and activates antioxidant gene expression through association with the AREs in the promoter region of the genes. KEAP1 represses NRF2 function in the nucleus by blocking NRF2 binding to AREs and exporting it to the cytoplasm for degradation. By competitively binding to KEAP1, PALB2 alleviates the negative impact of KEAP1 on NRF2 function via the two above-noted mechanisms. BRCA2 is also a component of the PALB2-KEAP1 complex, but its role is unclear.
Figure Legend Snippet: A proposed model of PALB2 regulation of NRF2 in the nucleus. Nuclear NRF2 forms heterodimers with small Maf proteins and activates antioxidant gene expression through association with the AREs in the promoter region of the genes. KEAP1 represses NRF2 function in the nucleus by blocking NRF2 binding to AREs and exporting it to the cytoplasm for degradation. By competitively binding to KEAP1, PALB2 alleviates the negative impact of KEAP1 on NRF2 function via the two above-noted mechanisms. BRCA2 is also a component of the PALB2-KEAP1 complex, but its role is unclear.

Techniques Used: Expressing, Blocking Assay, Binding Assay

PALB2 regulates NRF2 nuclear export after induction. (A) Western blotting of whole-cell extracts of U2OS cells (transfected with control or PALB2 siRNAs for 48 h) following treatment with DMSO or tBHQ and at indicated time points after tBHQ removal. (B) Results from 3 independent experiments in panel A were quantified by densitometry and plotted. (C) Western blotting of nuclear extracts of U2OS cells following the same treatments as those in panel A. (D) Results from 3 independent experiments in panel C were quantified by densitometry.
Figure Legend Snippet: PALB2 regulates NRF2 nuclear export after induction. (A) Western blotting of whole-cell extracts of U2OS cells (transfected with control or PALB2 siRNAs for 48 h) following treatment with DMSO or tBHQ and at indicated time points after tBHQ removal. (B) Results from 3 independent experiments in panel A were quantified by densitometry and plotted. (C) Western blotting of nuclear extracts of U2OS cells following the same treatments as those in panel A. (D) Results from 3 independent experiments in panel C were quantified by densitometry.

Techniques Used: Western Blot, Transfection

PALB2 overexpression promotes NRF2 nuclear accumulation and reduces ROS level. (A) An HA-NRF2 plasmid was cotransfected into 293T cells with empty Myc-GFP vector or Myc-PALB2WT-GFP or Myc-PALB2T92E-GFP constructs for 24 h, and their expression and localization were analyzed by fluorescence microscopy. (B) T98G cells overexpressing N-terminally FLAG-HA-double-tagged PALB2 were costained with HA (for PALB2) and NRF2 antibodies and visualized by fluorescence microscopy. (C) T98G cells in panel B were fractionated into cytosol and nuclei, and the amounts of indicated proteins in each compartment were analyzed by Western blotting. (D) Nuclei of HeLa cells harboring the vector or PALB2 overexpression construct were isolated, and protein amounts were analyzed by Western blotting. Lamin A/C, a nuclear protein, was used as a loading control. (E) ROS levels in naïve HeLa cells and the two stable cell lines in panel D were measured using the DCF assay. (F) U2OS cells were cotransfected with the ARE-Luc reporter system and various PALB2 expression vectors, and luciferase activities were measured 48 h after transfection. (G) Cells were transfected with the indicated expression vectors for 30 h, and the tagged PALB2 proteins were IPed with anti-FLAG M2 beads. The IPed PALB2 and co-IPed BRCA2, BRCA1, and KEAP1 proteins were probed with their respective antibodies by Western blotting.
Figure Legend Snippet: PALB2 overexpression promotes NRF2 nuclear accumulation and reduces ROS level. (A) An HA-NRF2 plasmid was cotransfected into 293T cells with empty Myc-GFP vector or Myc-PALB2WT-GFP or Myc-PALB2T92E-GFP constructs for 24 h, and their expression and localization were analyzed by fluorescence microscopy. (B) T98G cells overexpressing N-terminally FLAG-HA-double-tagged PALB2 were costained with HA (for PALB2) and NRF2 antibodies and visualized by fluorescence microscopy. (C) T98G cells in panel B were fractionated into cytosol and nuclei, and the amounts of indicated proteins in each compartment were analyzed by Western blotting. (D) Nuclei of HeLa cells harboring the vector or PALB2 overexpression construct were isolated, and protein amounts were analyzed by Western blotting. Lamin A/C, a nuclear protein, was used as a loading control. (E) ROS levels in naïve HeLa cells and the two stable cell lines in panel D were measured using the DCF assay. (F) U2OS cells were cotransfected with the ARE-Luc reporter system and various PALB2 expression vectors, and luciferase activities were measured 48 h after transfection. (G) Cells were transfected with the indicated expression vectors for 30 h, and the tagged PALB2 proteins were IPed with anti-FLAG M2 beads. The IPed PALB2 and co-IPed BRCA2, BRCA1, and KEAP1 proteins were probed with their respective antibodies by Western blotting.

Techniques Used: Over Expression, Plasmid Preparation, Construct, Expressing, Fluorescence, Microscopy, Western Blot, Isolation, Stable Transfection, DCF Assay, Luciferase, Transfection

siRNA-mediated depletion of PALB2 reduces NRF2 abundance and activity in the nucleus and increases cellular ROS level. (A) Western blotting showing the levels of proteins of interest after siRNA treatments. (B) ROS levels in cells depleted of each of the indicated proteins. (C) Endogenous NRF2 activity on an ARE-luciferase reporter following depletion of PALB2 and other proteins. (D and E) Nuclear NRF2 level after depletion of PALB2 in U2OS cells analyzed by Western blotting (D) and quantified by densitometry (E). (F and G) ChIP analysis of endogenous NRF2 binding to NQO1 ARE in control and PALB2-depleted cells. (F) An agarose gel image of the PCR products in a representative experiment. (G) Results quantified by real-time PCR.
Figure Legend Snippet: siRNA-mediated depletion of PALB2 reduces NRF2 abundance and activity in the nucleus and increases cellular ROS level. (A) Western blotting showing the levels of proteins of interest after siRNA treatments. (B) ROS levels in cells depleted of each of the indicated proteins. (C) Endogenous NRF2 activity on an ARE-luciferase reporter following depletion of PALB2 and other proteins. (D and E) Nuclear NRF2 level after depletion of PALB2 in U2OS cells analyzed by Western blotting (D) and quantified by densitometry (E). (F and G) ChIP analysis of endogenous NRF2 binding to NQO1 ARE in control and PALB2-depleted cells. (F) An agarose gel image of the PCR products in a representative experiment. (G) Results quantified by real-time PCR.

Techniques Used: Activity Assay, Western Blot, Luciferase, Chromatin Immunoprecipitation, Binding Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

8) Product Images from "Aldose reductase inhibitor, fidarestat regulates mitochondrial biogenesis via Nrf2/HO1/AMPK pathway in colon cancer cells"

Article Title: Aldose reductase inhibitor, fidarestat regulates mitochondrial biogenesis via Nrf2/HO1/AMPK pathway in colon cancer cells

Journal: Cancer letters

doi: 10.1016/j.canlet.2017.09.031

Effect of Fidarestat on Nrf2 and HO-1 expression in HT29 xenografts HT29 cells were injected subcutaneously into nude mouse and when tumors reached a cross-sectional area of about 30mm 2 ]. The xenograft tumor sections were immunostained with antibodies against Nrf2 and HO-1. The immunoreactivity of the antibody was assessed by visualizing the dark brown stain in the tumor cells, whereas the non-reactive areas displayed only the background color. Photomicrographs of the stained sections were acquired using EPI-800 microscope (bright-field) connected to a Nikon camera (×400 magnification). Images represent one of three independent analysis.
Figure Legend Snippet: Effect of Fidarestat on Nrf2 and HO-1 expression in HT29 xenografts HT29 cells were injected subcutaneously into nude mouse and when tumors reached a cross-sectional area of about 30mm 2 ]. The xenograft tumor sections were immunostained with antibodies against Nrf2 and HO-1. The immunoreactivity of the antibody was assessed by visualizing the dark brown stain in the tumor cells, whereas the non-reactive areas displayed only the background color. Photomicrographs of the stained sections were acquired using EPI-800 microscope (bright-field) connected to a Nikon camera (×400 magnification). Images represent one of three independent analysis.

Techniques Used: Expressing, Injection, Staining, Microscopy

Fidarestat induces Nrf2 downstream proteins such as HO-1, NQO1, and SOD in CRC cells The SW480 cells pre-treated with fidarestat (10μM) for 24 h followed by incubation with EGF (10 ng/ml) for an additional 24 h. ( A ) Equal amounts of cell extracts were subjected to Western blot analysis using specific antibodies against HO-1 and NQO1. Blots represent one of three independent analysis and antibodies against GAPDH were used as loading control. (B) . The levels of HO-1 in the cell lysates were determined by ELISA kit as per manufacturer’s instructions (Abcam). ( C ) The expression of HO-1 at the mRNA levels was determined by qPCR as described in the Methods section. (D) SOD activity was determined using ELISA kit according to the manufacturer’s instructions (Cayman Chemical). (E) Catalase activity was determined by spectrophotometric rate reduction of H 2 O 2 (units/ml enzyme) (Sigma -Aldrich). Data presented as mean ± SD (n=5). *p
Figure Legend Snippet: Fidarestat induces Nrf2 downstream proteins such as HO-1, NQO1, and SOD in CRC cells The SW480 cells pre-treated with fidarestat (10μM) for 24 h followed by incubation with EGF (10 ng/ml) for an additional 24 h. ( A ) Equal amounts of cell extracts were subjected to Western blot analysis using specific antibodies against HO-1 and NQO1. Blots represent one of three independent analysis and antibodies against GAPDH were used as loading control. (B) . The levels of HO-1 in the cell lysates were determined by ELISA kit as per manufacturer’s instructions (Abcam). ( C ) The expression of HO-1 at the mRNA levels was determined by qPCR as described in the Methods section. (D) SOD activity was determined using ELISA kit according to the manufacturer’s instructions (Cayman Chemical). (E) Catalase activity was determined by spectrophotometric rate reduction of H 2 O 2 (units/ml enzyme) (Sigma -Aldrich). Data presented as mean ± SD (n=5). *p

Techniques Used: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Activity Assay

Effect of AR inhibitor fidarestat on Nrf2 activation in CRC cells (A) SW480, HT29, and HCT 116 cells were treated with fidarestat (10 μM) for indicated times or pretreated with fidarestat for 24 h followed by incubation with EGF (10ng/mL) for an additional 30, 60, 120 and 240 mins. Equal amounts of nuclear proteins were subjected to Western blot analysis. (B) Nuclear localization of Nrf2 was determined by immunofluorescence of SW480 cells. The cells were immunostained with antibodies against Nrf2 followed by the addition of FITC (green)-labelled anti-rabbit antisera. DAPI (blue) staining was also performed to visualize the nuclei. The images shown are DAPI+FITC-labeled cells. (C) Nrf2-DNA binding activity was determined by ELISA in the nuclear extracts isolated from SW480 cells using a kit according to the manufacturer’s instructions (Cayman Chemical). Data presented as mean ± SD (n=5). *p
Figure Legend Snippet: Effect of AR inhibitor fidarestat on Nrf2 activation in CRC cells (A) SW480, HT29, and HCT 116 cells were treated with fidarestat (10 μM) for indicated times or pretreated with fidarestat for 24 h followed by incubation with EGF (10ng/mL) for an additional 30, 60, 120 and 240 mins. Equal amounts of nuclear proteins were subjected to Western blot analysis. (B) Nuclear localization of Nrf2 was determined by immunofluorescence of SW480 cells. The cells were immunostained with antibodies against Nrf2 followed by the addition of FITC (green)-labelled anti-rabbit antisera. DAPI (blue) staining was also performed to visualize the nuclei. The images shown are DAPI+FITC-labeled cells. (C) Nrf2-DNA binding activity was determined by ELISA in the nuclear extracts isolated from SW480 cells using a kit according to the manufacturer’s instructions (Cayman Chemical). Data presented as mean ± SD (n=5). *p

Techniques Used: Activation Assay, Incubation, Western Blot, Immunofluorescence, Staining, Labeling, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Isolation

Fidarestat-regulated cell viability of cancer cells is mediated by Nrf2 activation The untransfected, control and Nrf2-siRNA- transfectedSW480 cells were treated with fidarestat ± EGF for 24h and cell viability was determined by MTT assay. Data represent mean ± SD (n = 6). *p
Figure Legend Snippet: Fidarestat-regulated cell viability of cancer cells is mediated by Nrf2 activation The untransfected, control and Nrf2-siRNA- transfectedSW480 cells were treated with fidarestat ± EGF for 24h and cell viability was determined by MTT assay. Data represent mean ± SD (n = 6). *p

Techniques Used: Activation Assay, MTT Assay

9) Product Images from "The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells"

Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2015/187265

Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.
Figure Legend Snippet: Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

Techniques Used: Expressing

Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.
Figure Legend Snippet: Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.

Techniques Used: Expressing

Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.
Figure Legend Snippet: Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

Techniques Used:

10) Product Images from "Lipoxin A4 Preconditioning Attenuates Intestinal Ischemia Reperfusion Injury through Keap1/Nrf2 Pathway in a Lipoxin A4 Receptor Independent Manner"

Article Title: Lipoxin A4 Preconditioning Attenuates Intestinal Ischemia Reperfusion Injury through Keap1/Nrf2 Pathway in a Lipoxin A4 Receptor Independent Manner

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2016/9303606

Brusatol reversed the protective effects conferred by Lipoxin A4. Representative photomicrographs (400x) showing H E staining of intestine (a) and Chiu's score (b) was carried out to evaluate the injury degree; quantitative analysis using ELISA method was taken to assay the concentration of oxidative marker 15-F2t-Isoprostane (c) and SOD activity (d) in intestine mucosa. Representative Western blots and quantitative analyses showing total Keap1 (e) and HO-1 (g) protein and nuclear Nrf2 (f) protein expressions in intestine mucosa. Each bar represents the mean ± SEM ( n = 6 per group). ∗ p
Figure Legend Snippet: Brusatol reversed the protective effects conferred by Lipoxin A4. Representative photomicrographs (400x) showing H E staining of intestine (a) and Chiu's score (b) was carried out to evaluate the injury degree; quantitative analysis using ELISA method was taken to assay the concentration of oxidative marker 15-F2t-Isoprostane (c) and SOD activity (d) in intestine mucosa. Representative Western blots and quantitative analyses showing total Keap1 (e) and HO-1 (g) protein and nuclear Nrf2 (f) protein expressions in intestine mucosa. Each bar represents the mean ± SEM ( n = 6 per group). ∗ p

Techniques Used: Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Marker, Activity Assay, Western Blot

Proposed signaling mechanisms mediated by Lipoxin A4 in preventing intestinal epithelium cells from intestine ischemia reperfusion (IIR) injury. Lipoxin A4 promotes Keap1 dissociation from Nrf2 and leads to Nrf2 translocation from cytoplasm to nucleus, resulting in enhancing downstream HO-1 gene expression without binding to the Lipoxin A4 receptor (ALXR). Antioxidant enzyme HO-1 could possibly reduce cytochrome C release from mitochondria and decrease lipid peroxidative product 15-F2t-Isoprostane and elevate cell antioxidant ability evidenced by increase of superoxide dismutase (SOD) and finally decrease cells apoptosis. In addition, Lipoxin A4 binding to ALXR could also reduce damage of diamine oxidase (DAO) products, D-lactic acid (DLA), and intestinal-type fatty acid-binding protein (FABP2) release with other mechanisms.
Figure Legend Snippet: Proposed signaling mechanisms mediated by Lipoxin A4 in preventing intestinal epithelium cells from intestine ischemia reperfusion (IIR) injury. Lipoxin A4 promotes Keap1 dissociation from Nrf2 and leads to Nrf2 translocation from cytoplasm to nucleus, resulting in enhancing downstream HO-1 gene expression without binding to the Lipoxin A4 receptor (ALXR). Antioxidant enzyme HO-1 could possibly reduce cytochrome C release from mitochondria and decrease lipid peroxidative product 15-F2t-Isoprostane and elevate cell antioxidant ability evidenced by increase of superoxide dismutase (SOD) and finally decrease cells apoptosis. In addition, Lipoxin A4 binding to ALXR could also reduce damage of diamine oxidase (DAO) products, D-lactic acid (DLA), and intestinal-type fatty acid-binding protein (FABP2) release with other mechanisms.

Techniques Used: Translocation Assay, Expressing, Binding Assay

Effects of Lipoxin A4 on Keap1/Nrf2 pathway. Quantitative analysis using ELISA method was taken to assay the concentration of oxidative marker 15-F2t-Isoprostane (a) and SOD activity (b) in intestine mucosa. Representative Western blots and quantitative analyses showing total Keap1 (c) and HO-1 (d) protein and nuclear Nrf2 (e) protein expressions in intestine mucosa. Each bar represents the mean ± SEM ( n = 6 per group). ∗ p
Figure Legend Snippet: Effects of Lipoxin A4 on Keap1/Nrf2 pathway. Quantitative analysis using ELISA method was taken to assay the concentration of oxidative marker 15-F2t-Isoprostane (a) and SOD activity (b) in intestine mucosa. Representative Western blots and quantitative analyses showing total Keap1 (c) and HO-1 (d) protein and nuclear Nrf2 (e) protein expressions in intestine mucosa. Each bar represents the mean ± SEM ( n = 6 per group). ∗ p

Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Marker, Activity Assay, Western Blot

Effects of Lipoxin A4 and knockdown of Nrf2 on H/R-induced cell damage in IEC-6 intestinal epithelium cells. (a) Effects of gene knockdown by Nrf2 siRNA. (b) Relative lactate dehydrogenase (LDH) release of IEC-6 cells. (c) Determining nuclear Nrf2 expression by western blot. Bars are mean ± standard deviation from four independent experiments. ∗ p
Figure Legend Snippet: Effects of Lipoxin A4 and knockdown of Nrf2 on H/R-induced cell damage in IEC-6 intestinal epithelium cells. (a) Effects of gene knockdown by Nrf2 siRNA. (b) Relative lactate dehydrogenase (LDH) release of IEC-6 cells. (c) Determining nuclear Nrf2 expression by western blot. Bars are mean ± standard deviation from four independent experiments. ∗ p

Techniques Used: Expressing, Western Blot, Standard Deviation

11) Product Images from "A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling"

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

Journal: Nature

doi: 10.1038/s41586-018-0622-0

Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.
Figure Legend Snippet: Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

Techniques Used: Nuclear Magnetic Resonance, Labeling, Cell Culture, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Western Blot, Isolation, Incubation

Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.
Figure Legend Snippet: Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.

Techniques Used: Western Blot, Affinity Purification, Concentration Assay, Sample Prep, Expressing, Quantitative RT-PCR, shRNA, Incubation, SDS Page, Silver Staining, Purification

Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.
Figure Legend Snippet: Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.

Techniques Used: Molecular Weight, Western Blot, Expressing, Migration, Incubation, In Vitro, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitation Assay, Activity Assay, shRNA

Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.
Figure Legend Snippet: Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.

Techniques Used: Modification, Mutagenesis, Nuclear Magnetic Resonance, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Activation Assay

MS2 analysis of CR-MGx crosslinked KEAP1 peptide. a, Targeted Parallel reaction monitoring (PRM) transitions ( n =6). b, Annotated MS2 spectrum from the crosslinked C151-R135 KEAP1 peptide.
Figure Legend Snippet: MS2 analysis of CR-MGx crosslinked KEAP1 peptide. a, Targeted Parallel reaction monitoring (PRM) transitions ( n =6). b, Annotated MS2 spectrum from the crosslinked C151-R135 KEAP1 peptide.

Techniques Used:

12) Product Images from "Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma"

Article Title: Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma

Journal: American Journal of Cancer Research

doi:

Effects of ISL on Keap-1 and Nrf-2 on HepG2 cells. A. After various times of ISL treatment, expressions of Keap-1 and Nrf-2 mRNA were measured by RT-PCR. β-Actin was used as a standard. B. After various time treatments with ISL, expressions of Keap-1 and Nrf-2 proteins were measured by western blot. C. After various time treatments with ISL, ubiquitination of Nrf2 was measured by immunoprecipitation. All data are expressed as mean ± SEM from three independent experiments. * p
Figure Legend Snippet: Effects of ISL on Keap-1 and Nrf-2 on HepG2 cells. A. After various times of ISL treatment, expressions of Keap-1 and Nrf-2 mRNA were measured by RT-PCR. β-Actin was used as a standard. B. After various time treatments with ISL, expressions of Keap-1 and Nrf-2 proteins were measured by western blot. C. After various time treatments with ISL, ubiquitination of Nrf2 was measured by immunoprecipitation. All data are expressed as mean ± SEM from three independent experiments. * p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation

13) Product Images from "Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2"

Article Title: Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.16.7130-7139.2004

The BTB domain of Bach1 does not bind Cul3. Whole-cell extracts prepared from 293T cells transfected with expression plasmids (3 μg) in the combinations indicated were subjected to immunoprecipitation (IP) with anti-Flag antibody-conjugated beads followed by immunoblot (IB) analysis with an anti-Myc antibody (top). The expression level of each protein was monitored by immunoblot analysis with anti-Myc (middle) and anti-Flag (bottom) antibodies, respectively.
Figure Legend Snippet: The BTB domain of Bach1 does not bind Cul3. Whole-cell extracts prepared from 293T cells transfected with expression plasmids (3 μg) in the combinations indicated were subjected to immunoprecipitation (IP) with anti-Flag antibody-conjugated beads followed by immunoblot (IB) analysis with an anti-Myc antibody (top). The expression level of each protein was monitored by immunoblot analysis with anti-Myc (middle) and anti-Flag (bottom) antibodies, respectively.

Techniques Used: Transfection, Expressing, Immunoprecipitation

) was examined by immunoprecipitation (upper panel) as described above. The expression levels of Cys mutants were verified by immunoblot analysis with anti-Keap1 antibody (lower panel). (C) Schematic presentation of Cul3 deletion mutants. (D) The N-terminal sequence of Cul3 is crucial for its association with Keap1. Whole-cell extracts of 293T cells transfected with expression plasmids of Cul3 deletion mutants (1 μg) and Keap1 (1 μg) were prepared and subjected to immunoprecipitation with anti-Flag (M2) beads and immunoblotting with anti-Keap1 antibody. Analyses of cell lysates expressing 3xFlag Cul3 (lanes 1 and 3), N280 (lanes 4 and 5), or ΔN280 (lanes 6 and 7) in the presence (lanes 2, 3, 5, and 7) or absence (lanes 1, 4, and 6) of Keap1 are shown (top). The expression levels of Keap1 and Cul3 deletion mutants were verified by immunoblot analysis with anti-Keap1 and anti-Flag antibodies (middle and bottom, respectively)
Figure Legend Snippet: ) was examined by immunoprecipitation (upper panel) as described above. The expression levels of Cys mutants were verified by immunoblot analysis with anti-Keap1 antibody (lower panel). (C) Schematic presentation of Cul3 deletion mutants. (D) The N-terminal sequence of Cul3 is crucial for its association with Keap1. Whole-cell extracts of 293T cells transfected with expression plasmids of Cul3 deletion mutants (1 μg) and Keap1 (1 μg) were prepared and subjected to immunoprecipitation with anti-Flag (M2) beads and immunoblotting with anti-Keap1 antibody. Analyses of cell lysates expressing 3xFlag Cul3 (lanes 1 and 3), N280 (lanes 4 and 5), or ΔN280 (lanes 6 and 7) in the presence (lanes 2, 3, 5, and 7) or absence (lanes 1, 4, and 6) of Keap1 are shown (top). The expression levels of Keap1 and Cul3 deletion mutants were verified by immunoblot analysis with anti-Keap1 and anti-Flag antibodies (middle and bottom, respectively)

Techniques Used: Immunoprecipitation, Expressing, Sequencing, Transfection

Assay system to examine the degradation mechanism of Nrf2. (A) Keap1 promotes Nrf2 degradation in the in vivo degradation system. An Nrf2 expression vector (2 μg) was transfected into Cos7 cells (90% confluent) with or without the Keap1 expression vector (1.5 μg). At 24 h after transfection, the cells were treated with dimethyl sulfoxide (DMSO) (lanes 1, 2, 4, 6, and 8) and 2 μM MG132 (lanes 3, 5, 7, and 9) for 12 h and directly lysed in sodium dodecyl sulfate sample buffer. (Upper panel) Whole-cell extracts were subjected to immunoblot analysis with an anti-Nrf2 antibody. (Lower panel). The expression level of cotransfected EGFP was used as an internal control. (B) Proteasome-specific inhibitors stabilize the Nrf2 protein. Transfected cells were treated with DMSO (lane 4), 2 μM MG132 (lane 5), 2 μM clasto-lactacystin β-lactone (lane 6), and E64 (lane 7) for 12 h. Immunoblot analysis was performed as described above. (C and D) The Nrf2 expressed in this system was rapidly degraded in a Keap1-dependent manner. Nrf2 and ΔETGE mutant were transfected into cells along with Keap1. At 36 h after transfection, the cells were treated with 10 μM cycloheximide (CHX) per ml for the periods indicated. (Upper panel) Whole-cell extracts were subjected to immunoblot analysis with an anti-Nrf2 antibody. (Lower panel). The expression level of EGFP was used as an internal control. The averages of the relative band intensities of Nrf2 (open squares) and ΔETGE mutant (closed circles) represent two independent experiments performed in duplicate.
Figure Legend Snippet: Assay system to examine the degradation mechanism of Nrf2. (A) Keap1 promotes Nrf2 degradation in the in vivo degradation system. An Nrf2 expression vector (2 μg) was transfected into Cos7 cells (90% confluent) with or without the Keap1 expression vector (1.5 μg). At 24 h after transfection, the cells were treated with dimethyl sulfoxide (DMSO) (lanes 1, 2, 4, 6, and 8) and 2 μM MG132 (lanes 3, 5, 7, and 9) for 12 h and directly lysed in sodium dodecyl sulfate sample buffer. (Upper panel) Whole-cell extracts were subjected to immunoblot analysis with an anti-Nrf2 antibody. (Lower panel). The expression level of cotransfected EGFP was used as an internal control. (B) Proteasome-specific inhibitors stabilize the Nrf2 protein. Transfected cells were treated with DMSO (lane 4), 2 μM MG132 (lane 5), 2 μM clasto-lactacystin β-lactone (lane 6), and E64 (lane 7) for 12 h. Immunoblot analysis was performed as described above. (C and D) The Nrf2 expressed in this system was rapidly degraded in a Keap1-dependent manner. Nrf2 and ΔETGE mutant were transfected into cells along with Keap1. At 36 h after transfection, the cells were treated with 10 μM cycloheximide (CHX) per ml for the periods indicated. (Upper panel) Whole-cell extracts were subjected to immunoblot analysis with an anti-Nrf2 antibody. (Lower panel). The expression level of EGFP was used as an internal control. The averages of the relative band intensities of Nrf2 (open squares) and ΔETGE mutant (closed circles) represent two independent experiments performed in duplicate.

Techniques Used: In Vivo, Expressing, Plasmid Preparation, Transfection, Mutagenesis

Keap1 associates specifically with Cul3 in vivo and in vitro. (A) Complex formation of Keap1 and Cul3 in 293T cells. Endogenous Keap1 was precipitated with anti-Keap1 antibody and protein G beads (IP). The immunocomplex was subjected to immunoblot analysis with anti-Cul3 antibody. Whole-cell extracts of 293T cells expressing human Cul3 were used as a control (lanes 3 and 4). (B) Association between Keap1 and Cul3 in a transient-expression system. Whole-cell extracts prepared from 293T cells transfected with expression plasmids of HA-tagged Keap1 (1 μg) and 3xFlag Cul3 (1 μg) were subjected to immunoprecipitation (IP) with anti-Flag (M2) beads and immunoblot analysis with anti-HA antibody (IB). Analyses of cells expressing 3xFlag Cul3 with or without HA-Keap1 (lanes 1 and 2) are shown. Lane 3 is loaded with cell extracts expressing HA-Keap1 alone. (C) Among the Cul family proteins, Cul3 specifically interacts with Keap1. Expression plasmids (1 μg each) of Cul1 (lanes 1 and 6), Cul2 (lanes 2 and 7), Cul3 (lanes 3 and 8), Cul4A (lanes 4 and 9), and Cul5 (lanes 5 and 10) were transfected into 293T cells in the presence (lanes 1 to 5) or absence (lanes 6 to 10) of Flag-fused Keap1. Immunoprecipitation and immunoblot analyses were performed as described above (top). The asterisk indicates a nonspecific band. The expression levels of Cul proteins and Flag-Keap1 were verified by immunoblot analysis with anti-Myc and anti-Keap1 antibodies (middle and bottom, respectively)
Figure Legend Snippet: Keap1 associates specifically with Cul3 in vivo and in vitro. (A) Complex formation of Keap1 and Cul3 in 293T cells. Endogenous Keap1 was precipitated with anti-Keap1 antibody and protein G beads (IP). The immunocomplex was subjected to immunoblot analysis with anti-Cul3 antibody. Whole-cell extracts of 293T cells expressing human Cul3 were used as a control (lanes 3 and 4). (B) Association between Keap1 and Cul3 in a transient-expression system. Whole-cell extracts prepared from 293T cells transfected with expression plasmids of HA-tagged Keap1 (1 μg) and 3xFlag Cul3 (1 μg) were subjected to immunoprecipitation (IP) with anti-Flag (M2) beads and immunoblot analysis with anti-HA antibody (IB). Analyses of cells expressing 3xFlag Cul3 with or without HA-Keap1 (lanes 1 and 2) are shown. Lane 3 is loaded with cell extracts expressing HA-Keap1 alone. (C) Among the Cul family proteins, Cul3 specifically interacts with Keap1. Expression plasmids (1 μg each) of Cul1 (lanes 1 and 6), Cul2 (lanes 2 and 7), Cul3 (lanes 3 and 8), Cul4A (lanes 4 and 9), and Cul5 (lanes 5 and 10) were transfected into 293T cells in the presence (lanes 1 to 5) or absence (lanes 6 to 10) of Flag-fused Keap1. Immunoprecipitation and immunoblot analyses were performed as described above (top). The asterisk indicates a nonspecific band. The expression levels of Cul proteins and Flag-Keap1 were verified by immunoblot analysis with anti-Myc and anti-Keap1 antibodies (middle and bottom, respectively)

Techniques Used: In Vivo, In Vitro, Expressing, Transfection, Immunoprecipitation

Ubiquitination of Nrf2 by Keap1 and Cul3 in vivo. Nrf2 (1 μg) was expressed in 293T cells, along with several combinations of Keap1 (0.5 μg) and Cul3 (1.5 μg)-Roc1 (1 μg), as indicated in the figure, in the presence of His-tagged ubiquitin (HisUb; 1 μg). As a control, the ΔETGE mutant was also transfected. Whole-cell extracts were prepared and subjected to affinity purification with Ni 2+ resin. Precipitates (ppt) were eluted by boiling in sodium dodecyl sulfate sample buffer and subjected to immunoblot (IB) analysis (upper panel) with anti-Nrf2 antibody. The expression level of Nrf2 in the whole-cell extracts was also verified by immunoblot analysis with anti-Nrf2 antibody (lower panel).
Figure Legend Snippet: Ubiquitination of Nrf2 by Keap1 and Cul3 in vivo. Nrf2 (1 μg) was expressed in 293T cells, along with several combinations of Keap1 (0.5 μg) and Cul3 (1.5 μg)-Roc1 (1 μg), as indicated in the figure, in the presence of His-tagged ubiquitin (HisUb; 1 μg). As a control, the ΔETGE mutant was also transfected. Whole-cell extracts were prepared and subjected to affinity purification with Ni 2+ resin. Precipitates (ppt) were eluted by boiling in sodium dodecyl sulfate sample buffer and subjected to immunoblot (IB) analysis (upper panel) with anti-Nrf2 antibody. The expression level of Nrf2 in the whole-cell extracts was also verified by immunoblot analysis with anti-Nrf2 antibody (lower panel).

Techniques Used: In Vivo, Mutagenesis, Transfection, Affinity Purification, Expressing

14) Product Images from "Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress"

Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

Journal: Oncotarget

doi:

Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.
Figure Legend Snippet: Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.

Techniques Used: Activity Assay, Multiple Displacement Amplification, Marker, Positive Control, Binding Assay

Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p
Figure Legend Snippet: Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p

Techniques Used: Multiple Displacement Amplification, Transfection, Western Blot, Positive Control

15) Product Images from "Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis"

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis

Journal: Scientific Reports

doi: 10.1038/srep44769

Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
Figure Legend Snippet: Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.

Techniques Used: Expressing, Immunohistochemistry, Staining

The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.
Figure Legend Snippet: The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.

Techniques Used: Expressing, Immunofluorescence, Staining

16) Product Images from "Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis"

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis

Journal: Scientific Reports

doi: 10.1038/srep44769

Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
Figure Legend Snippet: Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.

Techniques Used: Expressing, Immunohistochemistry, Staining

The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.
Figure Legend Snippet: The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.

Techniques Used: Expressing, Immunofluorescence, Staining

17) Product Images from "Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway"

Article Title: Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway

Journal: Redox Biology

doi: 10.1016/j.redox.2018.07.002

Polydatin alleviates liver inflammation and lipid deposition in fructose-fed rats. Rats were fed 10% fructose drinking water (wt/vl) for 13 weeks and treated with polydtain (7.5, 15, 30 mg/kg) and pioglitazone (4 mg/kg) during the last 7 weeks. (A) qRT-PCT analysis of TXNIP mRNA levels, and (B) Western blot analysis of TXNIP protein levels in rat livers (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (C) Representative microphotograph of H E-stained and oil-red O-stained paraffin-embedded sections of liver tissues were shown (200 and 400× magnification; bars, 100 µm), respectively. (D) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in rat livers (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin alleviates liver inflammation and lipid deposition in fructose-fed rats. Rats were fed 10% fructose drinking water (wt/vl) for 13 weeks and treated with polydtain (7.5, 15, 30 mg/kg) and pioglitazone (4 mg/kg) during the last 7 weeks. (A) qRT-PCT analysis of TXNIP mRNA levels, and (B) Western blot analysis of TXNIP protein levels in rat livers (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (C) Representative microphotograph of H E-stained and oil-red O-stained paraffin-embedded sections of liver tissues were shown (200 and 400× magnification; bars, 100 µm), respectively. (D) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in rat livers (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Western Blot, Staining, Cycling Probe Technology

Polydatin inhibits ROS/TXNIP to reduce inflammation and lipid deposition in fructose-exposed BRL-3A cells. (A) Western blot analysis of TXNIP protein levels in 5 mM NAC treated BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (48 h) (n = 4 at least). Relative protein levels of TXNIP were normalized to GAPDH. (B) Assay of ROS levels (24 h, n = 7 at least), (C) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels (48 h) (n = 4 at least), and detection of TG (D) and TC (E) levels (48 h) (n = 4 at least) in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin inhibits ROS/TXNIP to reduce inflammation and lipid deposition in fructose-exposed BRL-3A cells. (A) Western blot analysis of TXNIP protein levels in 5 mM NAC treated BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (48 h) (n = 4 at least). Relative protein levels of TXNIP were normalized to GAPDH. (B) Assay of ROS levels (24 h, n = 7 at least), (C) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels (48 h) (n = 4 at least), and detection of TG (D) and TC (E) levels (48 h) (n = 4 at least) in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Western Blot, Incubation, Cycling Probe Technology, Transfection

Polydatin reduces fructose-induced oxidative stress, inflammation and lipid accumulation in BRL-3A and HepG2 cells. BRL-3A and HepG2 cells were cultured with or without 5 mM fructose in the presence or absence of polydatin (10, 20 and 40 μM) or pioglitazone (10 μM), respectively. (A) ROS levels were analyzed by labeling fluorogenic probe DCFH 2 -DA (n = 8). (B, C) qRT-PCT analysis of TXNIP mRNA levels and Western blot analysis of TXNIP protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (D, E) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. (F) IL-1β levels were detected by ELISA in the supernatant of BRL-3A and HepG2 cells (24 h) (n = 4 at least). (G) TG and TC levels were measured with standard diagnostic kits in BRL-3A and HepG2 cells (48 h) (n = 4 at least). All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin reduces fructose-induced oxidative stress, inflammation and lipid accumulation in BRL-3A and HepG2 cells. BRL-3A and HepG2 cells were cultured with or without 5 mM fructose in the presence or absence of polydatin (10, 20 and 40 μM) or pioglitazone (10 μM), respectively. (A) ROS levels were analyzed by labeling fluorogenic probe DCFH 2 -DA (n = 8). (B, C) qRT-PCT analysis of TXNIP mRNA levels and Western blot analysis of TXNIP protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (D, E) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. (F) IL-1β levels were detected by ELISA in the supernatant of BRL-3A and HepG2 cells (24 h) (n = 4 at least). (G) TG and TC levels were measured with standard diagnostic kits in BRL-3A and HepG2 cells (48 h) (n = 4 at least). All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Cell Culture, Labeling, Western Blot, Cycling Probe Technology, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

18) Product Images from "C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21"

Article Title: C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21

Journal: Diabetologia

doi: 10.1007/s00125-016-3958-8

Possible mechanisms for the prevention of diabetic nephropathy by C66. On one hand, C66 upregulates miR-200a to enhance NRF2 function by targeting Keap1 , leading to alleviation of renal oxidative damage. On the other hand, C66 inhibits miR-21-induced
Figure Legend Snippet: Possible mechanisms for the prevention of diabetic nephropathy by C66. On one hand, C66 upregulates miR-200a to enhance NRF2 function by targeting Keap1 , leading to alleviation of renal oxidative damage. On the other hand, C66 inhibits miR-21-induced

Techniques Used:

Both C66 and LNA-21 downregulate miR-21 in Nrf2 -null mice, resulting in reduced levels of stimulators for diabetic renal fibrosis. ( a , b ) In Nrf2 -null mice, expression levels of renal miR-21 ( a ) and Smad7 ( b ) RNA were evaluated by RT-PCR. ( c –
Figure Legend Snippet: Both C66 and LNA-21 downregulate miR-21 in Nrf2 -null mice, resulting in reduced levels of stimulators for diabetic renal fibrosis. ( a , b ) In Nrf2 -null mice, expression levels of renal miR-21 ( a ) and Smad7 ( b ) RNA were evaluated by RT-PCR. ( c –

Techniques Used: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Deletion of the Nrf2 gene partially abolished the renal protection afforded by C66 against diabetes-induced albuminuria. ( a , b ) Blood glucose levels in C57 WT mice ( a ) and Nrf2 -null mice ( b ) were monitored at 0, 4, 8, 12, 16, 20 and 24 weeks after diabetes
Figure Legend Snippet: Deletion of the Nrf2 gene partially abolished the renal protection afforded by C66 against diabetes-induced albuminuria. ( a , b ) Blood glucose levels in C57 WT mice ( a ) and Nrf2 -null mice ( b ) were monitored at 0, 4, 8, 12, 16, 20 and 24 weeks after diabetes

Techniques Used: Mouse Assay

C66 upregulation of renal NRF2 requires an increase in miR-200a. ( a – e ) The effects of C66, diabetes, LNA-200a and their combinations were compared on RNA levels of miR-200a ( a ) and Keap1 ( b ) and on protein levels of KEAP1 ( c ), total NRF2 ( d ) and
Figure Legend Snippet: C66 upregulation of renal NRF2 requires an increase in miR-200a. ( a – e ) The effects of C66, diabetes, LNA-200a and their combinations were compared on RNA levels of miR-200a ( a ) and Keap1 ( b ) and on protein levels of KEAP1 ( c ), total NRF2 ( d ) and

Techniques Used:

Both C66 and LNA-21 attenuated diabetes-induced pathological changes and albuminuria in the absence of NRF2. ( a – d ) In Nrf2 -null mice, PAS and Masson’s trichrome staining ( a ) were performed with glomerular area ( b ) and mesangial matrix
Figure Legend Snippet: Both C66 and LNA-21 attenuated diabetes-induced pathological changes and albuminuria in the absence of NRF2. ( a – d ) In Nrf2 -null mice, PAS and Masson’s trichrome staining ( a ) were performed with glomerular area ( b ) and mesangial matrix

Techniques Used: Mouse Assay, Staining

19) Product Images from "Extraction, identification, and antioxidant property evaluation of limonin from pummelo seeds"

Article Title: Extraction, identification, and antioxidant property evaluation of limonin from pummelo seeds

Journal: Animal Nutrition

doi: 10.1016/j.aninu.2018.05.005

Effect of limonin on the transcriptional (A, gene expressions) and posttranscriptional of Nrf2-ARE pathway (B, protein synthesis). Significance is marked by star bars. * stands for P
Figure Legend Snippet: Effect of limonin on the transcriptional (A, gene expressions) and posttranscriptional of Nrf2-ARE pathway (B, protein synthesis). Significance is marked by star bars. * stands for P

Techniques Used:

20) Product Images from "The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells"

Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2015/187265

Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.
Figure Legend Snippet: Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

Techniques Used: Expressing

Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.
Figure Legend Snippet: Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.

Techniques Used: Expressing

Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.
Figure Legend Snippet: Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

Techniques Used:

21) Product Images from "Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation"

Article Title: Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.322

HBx-stimulated p62–Keap1 interaction is required for HBx-induced Nrf2 activation. ( a ) Localization of Dsred-Keap1 and Nrf2 in HBx-GFP-transfected Huh7 cells. ( b ) Colocalization of Dsred-Keap1 and p62 in HBx-GFP-expressing Huh7 cells. ( c ) Co-immunoprecipitation of Keap1 with p62 (left panel) or p62 with Keap1 (right panel) in HBx-expressing cells. ( d ) Immunostaining of p62 and Keap1 in normal liver tissues and HBV-infected non-tumor or tumor tissues. ( e ) Co-immunoprecipitation of HBx-GFP, p62, and Keap1 in HBx-GFP-expressing cells transfected without or with p62 siRNAs, Flag-p62, or Flag-p62T352A (p62TA). ( f ) Localization of Dsred-Keap1 in HBx-GFP-transfected cells with Flag-p62TA expression (upper panel) or p62 RNAi (lower panel). ( g ) Nqo1 mRNA level measured by qRT-PCR in HBx-GFP-expressing cells with or without p62 RNAi. ( h ) G6PD enzyme activity in cells treated as in ( g ). All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P
Figure Legend Snippet: HBx-stimulated p62–Keap1 interaction is required for HBx-induced Nrf2 activation. ( a ) Localization of Dsred-Keap1 and Nrf2 in HBx-GFP-transfected Huh7 cells. ( b ) Colocalization of Dsred-Keap1 and p62 in HBx-GFP-expressing Huh7 cells. ( c ) Co-immunoprecipitation of Keap1 with p62 (left panel) or p62 with Keap1 (right panel) in HBx-expressing cells. ( d ) Immunostaining of p62 and Keap1 in normal liver tissues and HBV-infected non-tumor or tumor tissues. ( e ) Co-immunoprecipitation of HBx-GFP, p62, and Keap1 in HBx-GFP-expressing cells transfected without or with p62 siRNAs, Flag-p62, or Flag-p62T352A (p62TA). ( f ) Localization of Dsred-Keap1 in HBx-GFP-transfected cells with Flag-p62TA expression (upper panel) or p62 RNAi (lower panel). ( g ) Nqo1 mRNA level measured by qRT-PCR in HBx-GFP-expressing cells with or without p62 RNAi. ( h ) G6PD enzyme activity in cells treated as in ( g ). All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Expressing, Immunoprecipitation, Immunostaining, Infection, Quantitative RT-PCR, Activity Assay

HBx enhances G6PD activity via Nrf2. ( a and b ) Western blot ( a ) and qRT-PCR ( b ) analysis of G6PD expression in Huh7 cells expressing HBx-Myc. ( c ) The relative G6PD enzyme activity in cells treated as in ( a ). ( d – f ) The protein level ( d ), mRNA level ( e ), and enzyme activity ( f ) of G6PD in Huh7 cell with or without HBx-Myc expression and Nrf2 RNAi. All the statistical data are presented as mean±S.E.M. of triplicate experiments. * P
Figure Legend Snippet: HBx enhances G6PD activity via Nrf2. ( a and b ) Western blot ( a ) and qRT-PCR ( b ) analysis of G6PD expression in Huh7 cells expressing HBx-Myc. ( c ) The relative G6PD enzyme activity in cells treated as in ( a ). ( d – f ) The protein level ( d ), mRNA level ( e ), and enzyme activity ( f ) of G6PD in Huh7 cell with or without HBx-Myc expression and Nrf2 RNAi. All the statistical data are presented as mean±S.E.M. of triplicate experiments. * P

Techniques Used: Activity Assay, Western Blot, Quantitative RT-PCR, Expressing

HBV stimulates Nrf2 activation. ( a ) Immunostaining of Nrf2 in HepG2 and HepG2.2.15 cells. Note the nuclear distribution of Nrf2 in HepG2.2.15 cells. ( b ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in HepG2 and HepG2.2.15 cells. ( c ) Huh7 cells transfected with HBV genomic DNA (HBV) or HBx-negative HBV genomic DNA (HBVX − ) were stained with HBcAg and Nrf2 antibodies at 48 h after transfection. ( d ) Huh7 cells were either treated with 250 μ M H 2 O 2 for 4 h or transfected with GFP or HBx-GFP for 48 h. Then, the cells were fixed and stained with Nrf2 antibody. ( e ) Western blot analysis of Nrf2 protein in nuclear extracts (N-Nrf2) and cell lysis (T-Nrf2) in cells treated as in ( d ). Lamin B was used as an internal control of nuclear fraction. ( f ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in cells treated as in ( d ). ( g ) Nqo1 and GSTm1 mRNA levels in normal liver tissues and HBV-infected non-tumor or tumor tissues. All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P
Figure Legend Snippet: HBV stimulates Nrf2 activation. ( a ) Immunostaining of Nrf2 in HepG2 and HepG2.2.15 cells. Note the nuclear distribution of Nrf2 in HepG2.2.15 cells. ( b ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in HepG2 and HepG2.2.15 cells. ( c ) Huh7 cells transfected with HBV genomic DNA (HBV) or HBx-negative HBV genomic DNA (HBVX − ) were stained with HBcAg and Nrf2 antibodies at 48 h after transfection. ( d ) Huh7 cells were either treated with 250 μ M H 2 O 2 for 4 h or transfected with GFP or HBx-GFP for 48 h. Then, the cells were fixed and stained with Nrf2 antibody. ( e ) Western blot analysis of Nrf2 protein in nuclear extracts (N-Nrf2) and cell lysis (T-Nrf2) in cells treated as in ( d ). Lamin B was used as an internal control of nuclear fraction. ( f ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in cells treated as in ( d ). ( g ) Nqo1 and GSTm1 mRNA levels in normal liver tissues and HBV-infected non-tumor or tumor tissues. All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P

Techniques Used: Activation Assay, Immunostaining, Quantitative RT-PCR, Transfection, Staining, Western Blot, Lysis, Infection

Nrf2 and G6PD are required to HBx-promoted cell proliferation. ( a ) G6PD and Nrf2 protein levels in HBx-GFP cells with the expression of indicated shRNAs. ( b ) Cell proliferation assay of the cells in ( a ). Data are presented as mean±S.E.M. of triplicate experiments. ( c ) Colony forma tion assay of the cells in ( a ). Colonies in three randomly chosen fields per well were quantified. Data are presented as mean±S.E.M. of triplicate experiments. ** P
Figure Legend Snippet: Nrf2 and G6PD are required to HBx-promoted cell proliferation. ( a ) G6PD and Nrf2 protein levels in HBx-GFP cells with the expression of indicated shRNAs. ( b ) Cell proliferation assay of the cells in ( a ). Data are presented as mean±S.E.M. of triplicate experiments. ( c ) Colony forma tion assay of the cells in ( a ). Colonies in three randomly chosen fields per well were quantified. Data are presented as mean±S.E.M. of triplicate experiments. ** P

Techniques Used: Expressing, Proliferation Assay

22) Product Images from "Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway"

Article Title: Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway

Journal: Redox Biology

doi: 10.1016/j.redox.2018.07.002

Polydatin alleviates liver inflammation and lipid deposition in fructose-fed rats. Rats were fed 10% fructose drinking water (wt/vl) for 13 weeks and treated with polydtain (7.5, 15, 30 mg/kg) and pioglitazone (4 mg/kg) during the last 7 weeks. (A) qRT-PCT analysis of TXNIP mRNA levels, and (B) Western blot analysis of TXNIP protein levels in rat livers (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (C) Representative microphotograph of H E-stained and oil-red O-stained paraffin-embedded sections of liver tissues were shown (200 and 400× magnification; bars, 100 µm), respectively. (D) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in rat livers (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin alleviates liver inflammation and lipid deposition in fructose-fed rats. Rats were fed 10% fructose drinking water (wt/vl) for 13 weeks and treated with polydtain (7.5, 15, 30 mg/kg) and pioglitazone (4 mg/kg) during the last 7 weeks. (A) qRT-PCT analysis of TXNIP mRNA levels, and (B) Western blot analysis of TXNIP protein levels in rat livers (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (C) Representative microphotograph of H E-stained and oil-red O-stained paraffin-embedded sections of liver tissues were shown (200 and 400× magnification; bars, 100 µm), respectively. (D) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in rat livers (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Western Blot, Staining, Cycling Probe Technology

Polydatin inhibits ROS/TXNIP to reduce inflammation and lipid deposition in fructose-exposed BRL-3A cells. (A) Western blot analysis of TXNIP protein levels in 5 mM NAC treated BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (48 h) (n = 4 at least). Relative protein levels of TXNIP were normalized to GAPDH. (B) Assay of ROS levels (24 h, n = 7 at least), (C) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels (48 h) (n = 4 at least), and detection of TG (D) and TC (E) levels (48 h) (n = 4 at least) in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin inhibits ROS/TXNIP to reduce inflammation and lipid deposition in fructose-exposed BRL-3A cells. (A) Western blot analysis of TXNIP protein levels in 5 mM NAC treated BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (48 h) (n = 4 at least). Relative protein levels of TXNIP were normalized to GAPDH. (B) Assay of ROS levels (24 h, n = 7 at least), (C) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels (48 h) (n = 4 at least), and detection of TG (D) and TC (E) levels (48 h) (n = 4 at least) in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Western Blot, Incubation, Cycling Probe Technology, Transfection

Polydatin reduces fructose-induced oxidative stress, inflammation and lipid accumulation in BRL-3A and HepG2 cells. BRL-3A and HepG2 cells were cultured with or without 5 mM fructose in the presence or absence of polydatin (10, 20 and 40 μM) or pioglitazone (10 μM), respectively. (A) ROS levels were analyzed by labeling fluorogenic probe DCFH 2 -DA (n = 8). (B, C) qRT-PCT analysis of TXNIP mRNA levels and Western blot analysis of TXNIP protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (D, E) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. (F) IL-1β levels were detected by ELISA in the supernatant of BRL-3A and HepG2 cells (24 h) (n = 4 at least). (G) TG and TC levels were measured with standard diagnostic kits in BRL-3A and HepG2 cells (48 h) (n = 4 at least). All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin reduces fructose-induced oxidative stress, inflammation and lipid accumulation in BRL-3A and HepG2 cells. BRL-3A and HepG2 cells were cultured with or without 5 mM fructose in the presence or absence of polydatin (10, 20 and 40 μM) or pioglitazone (10 μM), respectively. (A) ROS levels were analyzed by labeling fluorogenic probe DCFH 2 -DA (n = 8). (B, C) qRT-PCT analysis of TXNIP mRNA levels and Western blot analysis of TXNIP protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (D, E) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. (F) IL-1β levels were detected by ELISA in the supernatant of BRL-3A and HepG2 cells (24 h) (n = 4 at least). (G) TG and TC levels were measured with standard diagnostic kits in BRL-3A and HepG2 cells (48 h) (n = 4 at least). All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Cell Culture, Labeling, Western Blot, Cycling Probe Technology, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

23) Product Images from "Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa"

Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

Journal: Scientific Reports

doi: 10.1038/s41598-018-30562-y

MCM3 and Nrf2 bind to Keap1 in structurally highly similar and competitive manner. ( a ) Sequence alignment of the H2I beta hairpin motifs from human MCM2-7 and Sulfolobus solfataricus (Sso) MCM proteins. ( b ) A cartoon showing the conserved order of MCM subunits in MCM2-7 heterohexamer and H2I hairpins in the central channel. ( c ) Structure models of Saccharomyces cerevisiae ). Kelch domain (beige) is viewed from the side opposite to the binding pocket. MCM2-7 is shown as a top view on its N-terminal tier, MCM3 subunit coloured light blue and opposite MCM6 subunit green. The Keap1 interacting beta hairpin motifs of MCM3 and Nrf2 proteins are in dark blue and marked by boxes here and on panel ‘d’, with ETGE box residues presented by red sphere models. ( d ) Side view (horizontal clockwise 90° rotation) of the same models, where all the other MCM subunits apart from MCM3 and MCM6 have been removed to reveal the central channel of MCM2-7 ring. ( e for images of full-length blots. ( f for images of full-length blots.
Figure Legend Snippet: MCM3 and Nrf2 bind to Keap1 in structurally highly similar and competitive manner. ( a ) Sequence alignment of the H2I beta hairpin motifs from human MCM2-7 and Sulfolobus solfataricus (Sso) MCM proteins. ( b ) A cartoon showing the conserved order of MCM subunits in MCM2-7 heterohexamer and H2I hairpins in the central channel. ( c ) Structure models of Saccharomyces cerevisiae ). Kelch domain (beige) is viewed from the side opposite to the binding pocket. MCM2-7 is shown as a top view on its N-terminal tier, MCM3 subunit coloured light blue and opposite MCM6 subunit green. The Keap1 interacting beta hairpin motifs of MCM3 and Nrf2 proteins are in dark blue and marked by boxes here and on panel ‘d’, with ETGE box residues presented by red sphere models. ( d ) Side view (horizontal clockwise 90° rotation) of the same models, where all the other MCM subunits apart from MCM3 and MCM6 have been removed to reveal the central channel of MCM2-7 ring. ( e for images of full-length blots. ( f for images of full-length blots.

Techniques Used: Sequencing, Binding Assay

24) Product Images from "Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa"

Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

Journal: Scientific Reports

doi: 10.1038/s41598-018-30562-y

Keap1, MCM3, and MCM-BP form a ternary complex. ( a for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
Figure Legend Snippet: Keap1, MCM3, and MCM-BP form a ternary complex. ( a for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.

Techniques Used: Affinity Purification, Infection, Expressing, Staining, SDS Page, Size-exclusion Chromatography, Co-Elution Assay, Molecular Weight, Mass Spectrometry

MCM3 and Nrf2 bind to Keap1 in structurally highly similar and competitive manner. ( a ) Sequence alignment of the H2I beta hairpin motifs from human MCM2-7 and Sulfolobus solfataricus (Sso) MCM proteins. ( b ) A cartoon showing the conserved order of MCM subunits in MCM2-7 heterohexamer and H2I hairpins in the central channel. ( c ) Structure models of Saccharomyces cerevisiae ). Kelch domain (beige) is viewed from the side opposite to the binding pocket. MCM2-7 is shown as a top view on its N-terminal tier, MCM3 subunit coloured light blue and opposite MCM6 subunit green. The Keap1 interacting beta hairpin motifs of MCM3 and Nrf2 proteins are in dark blue and marked by boxes here and on panel ‘d’, with ETGE box residues presented by red sphere models. ( d ) Side view (horizontal clockwise 90° rotation) of the same models, where all the other MCM subunits apart from MCM3 and MCM6 have been removed to reveal the central channel of MCM2-7 ring. ( e for images of full-length blots. ( f for images of full-length blots.
Figure Legend Snippet: MCM3 and Nrf2 bind to Keap1 in structurally highly similar and competitive manner. ( a ) Sequence alignment of the H2I beta hairpin motifs from human MCM2-7 and Sulfolobus solfataricus (Sso) MCM proteins. ( b ) A cartoon showing the conserved order of MCM subunits in MCM2-7 heterohexamer and H2I hairpins in the central channel. ( c ) Structure models of Saccharomyces cerevisiae ). Kelch domain (beige) is viewed from the side opposite to the binding pocket. MCM2-7 is shown as a top view on its N-terminal tier, MCM3 subunit coloured light blue and opposite MCM6 subunit green. The Keap1 interacting beta hairpin motifs of MCM3 and Nrf2 proteins are in dark blue and marked by boxes here and on panel ‘d’, with ETGE box residues presented by red sphere models. ( d ) Side view (horizontal clockwise 90° rotation) of the same models, where all the other MCM subunits apart from MCM3 and MCM6 have been removed to reveal the central channel of MCM2-7 ring. ( e for images of full-length blots. ( f for images of full-length blots.

Techniques Used: Sequencing, Binding Assay

Helix-2 insert (H2I) hairpin and its conserved ETGE sequence box are required for normal growth and minichromosome maintenance function of MCM3 in yeast. ( a ) Tetrad dissection of yeast strains that carry mcm3 alleles with the mutations in H2I motif as depicted schematically on the right (dashed lines correspond to deleted regions). For each MCM3/mcm3 diploid strain, two tetrads are shown that were grown for three days after dissection. Arrowheads indicate clones with a mutated allele. ( b ) Competitive co-growth of wild type (WT) yeast with strains carrying mcm3 - GAGA (upper panel) or mcm3 - del449 -454 (lower panel) mutant alleles. The WT and mutant strains were pre-grown separately before mixing together on day 0 and co-growing for four days. Genomic DNA from a resulting co-culture was analysed by PCR and following restriction analysis with AluI ( mcm3 - GAGA ) or XhoI ( mcm3 - del449 - 454 ), which cleave the mutant but not WT DNA fragment. ( c ) Plasmid minichromosome maintenance assay with mcm3 - GAGA and mcm3 - del449 - 454 strains. Cells were transformed with pRS416 plasmid and grown for four days without selection, determining the percentage of plasmid-carrying cells each day.
Figure Legend Snippet: Helix-2 insert (H2I) hairpin and its conserved ETGE sequence box are required for normal growth and minichromosome maintenance function of MCM3 in yeast. ( a ) Tetrad dissection of yeast strains that carry mcm3 alleles with the mutations in H2I motif as depicted schematically on the right (dashed lines correspond to deleted regions). For each MCM3/mcm3 diploid strain, two tetrads are shown that were grown for three days after dissection. Arrowheads indicate clones with a mutated allele. ( b ) Competitive co-growth of wild type (WT) yeast with strains carrying mcm3 - GAGA (upper panel) or mcm3 - del449 -454 (lower panel) mutant alleles. The WT and mutant strains were pre-grown separately before mixing together on day 0 and co-growing for four days. Genomic DNA from a resulting co-culture was analysed by PCR and following restriction analysis with AluI ( mcm3 - GAGA ) or XhoI ( mcm3 - del449 - 454 ), which cleave the mutant but not WT DNA fragment. ( c ) Plasmid minichromosome maintenance assay with mcm3 - GAGA and mcm3 - del449 - 454 strains. Cells were transformed with pRS416 plasmid and grown for four days without selection, determining the percentage of plasmid-carrying cells each day.

Techniques Used: Sequencing, Dissection, Clone Assay, Mutagenesis, Co-Culture Assay, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay, Selection

siRNA knock-down of MCM3 levels results in lower sensitivity of Keap1 - Nrf2 response. ( a ) Western blotting analysis of human U2OS cells transfected with MCM3 siRNA #1, or negative control siRNA, and treated with indicated concentrations of tBHQ to induce the Keap1 controlled stabilization of Nrf2 protein. MCM3 blot shows the efficiency of a knock-down and actin blot serves as a loading control in all the panels of this figure. ( b ) Similar experiment, where different siRNA was used (#2) to knock down the MCM3 expression, and cells were treated with higher tBHQ concentrations. Nrf2 transactivation target heme oxygenase 1 (HO1) was additionally blotted. ( c ) The knock-down experiment with MCM3 siRNA #1, where different chemical activator (DEM) was used to induce the Keap1 controlled Nrf2 response. ( d ) Transfection experiments with U2OS cells showing the induction of Nrf2 levels in response to 50 µM DEM treatment (6 hrs) in cells over-expressing either WT or ETGE > GAGA mutant MCM3. Ectopically expressed MCM3 carried N-terminal FLAG and MBP tags and was blotted using antibodies against the FLAG tag of the protein.
Figure Legend Snippet: siRNA knock-down of MCM3 levels results in lower sensitivity of Keap1 - Nrf2 response. ( a ) Western blotting analysis of human U2OS cells transfected with MCM3 siRNA #1, or negative control siRNA, and treated with indicated concentrations of tBHQ to induce the Keap1 controlled stabilization of Nrf2 protein. MCM3 blot shows the efficiency of a knock-down and actin blot serves as a loading control in all the panels of this figure. ( b ) Similar experiment, where different siRNA was used (#2) to knock down the MCM3 expression, and cells were treated with higher tBHQ concentrations. Nrf2 transactivation target heme oxygenase 1 (HO1) was additionally blotted. ( c ) The knock-down experiment with MCM3 siRNA #1, where different chemical activator (DEM) was used to induce the Keap1 controlled Nrf2 response. ( d ) Transfection experiments with U2OS cells showing the induction of Nrf2 levels in response to 50 µM DEM treatment (6 hrs) in cells over-expressing either WT or ETGE > GAGA mutant MCM3. Ectopically expressed MCM3 carried N-terminal FLAG and MBP tags and was blotted using antibodies against the FLAG tag of the protein.

Techniques Used: Western Blot, Transfection, Negative Control, Expressing, Mutagenesis, FLAG-tag

Characterisation of Keap1-MCM3 interaction. ( a for images of full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels of FLAG-MCM3 – strep-Keap1 tandem affinity pulldown (left panel), and strep-Keap1 – FLAG-MCM3 tandem affinity pull down (right panel) from the baculovirus infected Sf9 cells expressing mouse Keap1 and all six MCM2-7 subunit proteins. Lanes correspond to the eluted material from both pulldown steps and to the unbound material (‘flow’) from the second step as indicated.
Figure Legend Snippet: Characterisation of Keap1-MCM3 interaction. ( a for images of full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels of FLAG-MCM3 – strep-Keap1 tandem affinity pulldown (left panel), and strep-Keap1 – FLAG-MCM3 tandem affinity pull down (right panel) from the baculovirus infected Sf9 cells expressing mouse Keap1 and all six MCM2-7 subunit proteins. Lanes correspond to the eluted material from both pulldown steps and to the unbound material (‘flow’) from the second step as indicated.

Techniques Used: Staining, SDS Page, Infection, Expressing, Flow Cytometry

Comparative evolutionary sequence analysis of the DxETGE interaction box in MCM3, Nrf2, and Nrf1 proteins. Sequence homology alignment of DxETGE interaction box and its beta hairpin context in the proteins from indicated species. Black vertical line between MCM3 and Nrf1 columns indicates the presence of Keap1 orthologue in the respective species.
Figure Legend Snippet: Comparative evolutionary sequence analysis of the DxETGE interaction box in MCM3, Nrf2, and Nrf1 proteins. Sequence homology alignment of DxETGE interaction box and its beta hairpin context in the proteins from indicated species. Black vertical line between MCM3 and Nrf1 columns indicates the presence of Keap1 orthologue in the respective species.

Techniques Used: Sequencing

Keap1 interacts with MCM3 in mammalian cells. ( a for full-length blots. ( b for full-length gels and blots. ( c ) Proximity ligation analysis (PLA) of the Keap1 - MCM3 interaction in human primary epithelial keratinocytes (HPEK). The images of red PLA channel alone are shown in the left column, and combined with blue DAPI staining of nuclei in the right column. ‘Keap1 + MCM3’ indicates the images with interaction specific signals, other images correspond to the control experiments with single antibodies. Shown are the maximum intensity projection images of the Z stacks from confocal microscopy; white scale bar = 10 µM. ( d ) Scatter dot plot of the quantified data of nuclear and cytoplasmic Keap1 + MCM3 PLA signals (M3 + K1) compared to negative control with MCM3 antibody alone (M3). Each data point represents an average number of nuclear or cytoplasmic PLA dots per cell from one micrograph. Bars represent the mean and standard deviation of combined data from two independent PLA experiments, one slide analysed in first and two in second experiment and three different micrographs quantified from each slide. The significance values (***p
Figure Legend Snippet: Keap1 interacts with MCM3 in mammalian cells. ( a for full-length blots. ( b for full-length gels and blots. ( c ) Proximity ligation analysis (PLA) of the Keap1 - MCM3 interaction in human primary epithelial keratinocytes (HPEK). The images of red PLA channel alone are shown in the left column, and combined with blue DAPI staining of nuclei in the right column. ‘Keap1 + MCM3’ indicates the images with interaction specific signals, other images correspond to the control experiments with single antibodies. Shown are the maximum intensity projection images of the Z stacks from confocal microscopy; white scale bar = 10 µM. ( d ) Scatter dot plot of the quantified data of nuclear and cytoplasmic Keap1 + MCM3 PLA signals (M3 + K1) compared to negative control with MCM3 antibody alone (M3). Each data point represents an average number of nuclear or cytoplasmic PLA dots per cell from one micrograph. Bars represent the mean and standard deviation of combined data from two independent PLA experiments, one slide analysed in first and two in second experiment and three different micrographs quantified from each slide. The significance values (***p

Techniques Used: Ligation, Proximity Ligation Assay, Staining, Confocal Microscopy, Negative Control, Standard Deviation

25) Product Images from "Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway"

Article Title: Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway

Journal: Redox Biology

doi: 10.1016/j.redox.2018.07.002

The hypothetical mechanisms by which polydatin prevents fructose-induced in liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway. Fructose-induced miR-200a low-expression increased Keap1 to inhibit Nrf2 antioxidant pathway, and then caused ROS-driven TXNIP to promote NLRP3 inflammasome activation and lipid metabolism-related protein dysregulation, resulting in liver inflammation and lipid deposition. Polydatin protected fructose-induced liver inflammation and lipid deposition by which increased miR-200a expression to decrease Keap1 and activate Nrf2 antioxidant pathway, and then blocked ROS-driven TXNIP to suppress NLRP3 inflammasome activation and regulated lipid metabolism-related proteins.
Figure Legend Snippet: The hypothetical mechanisms by which polydatin prevents fructose-induced in liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway. Fructose-induced miR-200a low-expression increased Keap1 to inhibit Nrf2 antioxidant pathway, and then caused ROS-driven TXNIP to promote NLRP3 inflammasome activation and lipid metabolism-related protein dysregulation, resulting in liver inflammation and lipid deposition. Polydatin protected fructose-induced liver inflammation and lipid deposition by which increased miR-200a expression to decrease Keap1 and activate Nrf2 antioxidant pathway, and then blocked ROS-driven TXNIP to suppress NLRP3 inflammasome activation and regulated lipid metabolism-related proteins.

Techniques Used: Expressing, Activation Assay

Polydatin reduces fructose-induced oxidative stress, inflammation and lipid accumulation in BRL-3A and HepG2 cells. BRL-3A and HepG2 cells were cultured with or without 5 mM fructose in the presence or absence of polydatin (10, 20 and 40 μM) or pioglitazone (10 μM), respectively. (A) ROS levels were analyzed by labeling fluorogenic probe DCFH 2 -DA (n = 8). (B, C) qRT-PCT analysis of TXNIP mRNA levels and Western blot analysis of TXNIP protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (D, E) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. (F) IL-1β levels were detected by ELISA in the supernatant of BRL-3A and HepG2 cells (24 h) (n = 4 at least). (G) TG and TC levels were measured with standard diagnostic kits in BRL-3A and HepG2 cells (48 h) (n = 4 at least). All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin reduces fructose-induced oxidative stress, inflammation and lipid accumulation in BRL-3A and HepG2 cells. BRL-3A and HepG2 cells were cultured with or without 5 mM fructose in the presence or absence of polydatin (10, 20 and 40 μM) or pioglitazone (10 μM), respectively. (A) ROS levels were analyzed by labeling fluorogenic probe DCFH 2 -DA (n = 8). (B, C) qRT-PCT analysis of TXNIP mRNA levels and Western blot analysis of TXNIP protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative mRNA levels of TXNIP were normalized to β-actin. Relative protein levels of TXNIP were normalized to GAPDH. (D, E) Western blot analysis of NLRP3, ASC, Caspase-1, IL-1β, PPAR-α, CPT-1, SREBP-1 and SCD-1 protein levels in BRL-3A and HepG2 cells (48 h) (n = 4 at least). Relative protein levels of Caspase-1 were normalized to pro-Caspase-1, of IL-1β were normalized to pro-IL-1β, of TXNIP, NLRP3, ASC, PPAR-α, CPT-1, SREBP-1 and SCD-1 were normalized to GAPDH or β-actin, respectively. (F) IL-1β levels were detected by ELISA in the supernatant of BRL-3A and HepG2 cells (24 h) (n = 4 at least). (G) TG and TC levels were measured with standard diagnostic kits in BRL-3A and HepG2 cells (48 h) (n = 4 at least). All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Cell Culture, Labeling, Western Blot, Cycling Probe Technology, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

Polydatin activates Nrf2 antioxidant pathway to inhibit oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A, B) Western blot analysis of total and nuclear Nrf2 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (C, D) Western blot analysis of GST, HO-1 and NQO1 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (E) Western blot analysis of nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (F) assay of ROS levels (24 h) (n = 5 at laest), (G) Western blot analysis of TXNIP protein levels (48 h) (n = 4 at least) in 10 μM tBHQ pretreated-BRL-3A cells for 8 h incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. (H) Western blot analysis of nuclear Nrf2 protein levels in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (24 h) (n = 4 at least). Relative protein levels of nuclear Nrf2 were normalized to LaminA, of total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
Figure Legend Snippet: Polydatin activates Nrf2 antioxidant pathway to inhibit oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A, B) Western blot analysis of total and nuclear Nrf2 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (C, D) Western blot analysis of GST, HO-1 and NQO1 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (E) Western blot analysis of nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (F) assay of ROS levels (24 h) (n = 5 at laest), (G) Western blot analysis of TXNIP protein levels (48 h) (n = 4 at least) in 10 μM tBHQ pretreated-BRL-3A cells for 8 h incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. (H) Western blot analysis of nuclear Nrf2 protein levels in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (24 h) (n = 4 at least). Relative protein levels of nuclear Nrf2 were normalized to LaminA, of total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

Techniques Used: Western Blot, Incubation, Transfection

26) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Western Blot, Infection, Expressing, MTT Assay

27) Product Images from "Zeaxanthin induces Nrf2-mediated phase II enzymes in protection of cell death"

Article Title: Zeaxanthin induces Nrf2-mediated phase II enzymes in protection of cell death

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.190

GSH is the critical effector of the protective effects of Zea. Total GSH levels were measured following the Zea treatment of ARPE-19 cells: ( a ) dose response—cells were treated with 1, 10, or 50 μ M Zea for 24 h; ( b ) time response—cells were treated with 10 μ M Zea for 6, 12, 24 or 48 h; ( c ) Nrf2 knockdown response—cells were transfected with Nrf2 siRNA for 24 h, followed by treatment with 10 μ M Zea for an additional 24 h; ( d ) t -BHP response—cells were treated with 10 μ M Zea for 24 h, followed by challenge with 300 μ M t -BHP for an additional 6 h. In the presence of 50 μ M BSO, cells were treated with 10 μ M Zea for 24 h and then challenged with 300 μ M t -BHP for an additional 6 h, after which cell viability ( e ), the mitochondrial membrane potential ( f ), and apoptosis activation ( g ) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (* P
Figure Legend Snippet: GSH is the critical effector of the protective effects of Zea. Total GSH levels were measured following the Zea treatment of ARPE-19 cells: ( a ) dose response—cells were treated with 1, 10, or 50 μ M Zea for 24 h; ( b ) time response—cells were treated with 10 μ M Zea for 6, 12, 24 or 48 h; ( c ) Nrf2 knockdown response—cells were transfected with Nrf2 siRNA for 24 h, followed by treatment with 10 μ M Zea for an additional 24 h; ( d ) t -BHP response—cells were treated with 10 μ M Zea for 24 h, followed by challenge with 300 μ M t -BHP for an additional 6 h. In the presence of 50 μ M BSO, cells were treated with 10 μ M Zea for 24 h and then challenged with 300 μ M t -BHP for an additional 6 h, after which cell viability ( e ), the mitochondrial membrane potential ( f ), and apoptosis activation ( g ) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (* P

Techniques Used: Transfection, Activation Assay

Zea activates phase II enzymes through Nrf2 nuclear translocation. ARPE-19 cells were transiently transfected with Nrf2 siRNA at 100 pmol per well in six-well plates for 24 h, followed by treatment with 10 μ M Zea for 6 or 24 h. The mRNA levels of Nrf2 ( a ), HO-1 ( b ), and NQO-1 ( c ) were analyzed after 6 h of Zea treatment, and the protein levels of HO-1 and NQO-1 were analyzed after 24 h of Zea treatment ( d ). Protein levels of Nrf2 and Keap1 response to Zea dose-dependent and time-dependent effects were analyzed by western blot ( e and f ). Cyto- and nuclear Nrf2 levels after treatment with 10 μ M Zea for 0.5, 2, or 6 h ( g ). Co-immunoprecipitation assays after 6 h of treatment with 10 μ M Zea, antibodies against Nrf2 ( h ) and Keap1 ( i ) were used for immunoprecipitation, and the antibodies employed in the western blot analysis are indicated on the left side of the panel. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (* P
Figure Legend Snippet: Zea activates phase II enzymes through Nrf2 nuclear translocation. ARPE-19 cells were transiently transfected with Nrf2 siRNA at 100 pmol per well in six-well plates for 24 h, followed by treatment with 10 μ M Zea for 6 or 24 h. The mRNA levels of Nrf2 ( a ), HO-1 ( b ), and NQO-1 ( c ) were analyzed after 6 h of Zea treatment, and the protein levels of HO-1 and NQO-1 were analyzed after 24 h of Zea treatment ( d ). Protein levels of Nrf2 and Keap1 response to Zea dose-dependent and time-dependent effects were analyzed by western blot ( e and f ). Cyto- and nuclear Nrf2 levels after treatment with 10 μ M Zea for 0.5, 2, or 6 h ( g ). Co-immunoprecipitation assays after 6 h of treatment with 10 μ M Zea, antibodies against Nrf2 ( h ) and Keap1 ( i ) were used for immunoprecipitation, and the antibodies employed in the western blot analysis are indicated on the left side of the panel. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (* P

Techniques Used: Translocation Assay, Transfection, Western Blot, Immunoprecipitation

Possible mechanism of Zea protection against t -BHP-induced cell apoptosis. Zea could time and dose dependently induce expression of phase II enzymes through promoting Nrf2 nuclear translocation. GSH, the production of γ -GCL, was thereby increased, and accounted for anti-apoptotic effect of Zea. Meanwhile, the activation of PI3K/Akt pathway was found to work as upstream kinase regulating phase II enzymes expression and GSH production
Figure Legend Snippet: Possible mechanism of Zea protection against t -BHP-induced cell apoptosis. Zea could time and dose dependently induce expression of phase II enzymes through promoting Nrf2 nuclear translocation. GSH, the production of γ -GCL, was thereby increased, and accounted for anti-apoptotic effect of Zea. Meanwhile, the activation of PI3K/Akt pathway was found to work as upstream kinase regulating phase II enzymes expression and GSH production

Techniques Used: Expressing, Translocation Assay, Activation Assay

Zea activates the Nrf2/Keap1 pathway through PI3/Akt activation. ARPE-19 cells were treated with 10 μ M Zea for the indicated time, and kinase activation was analyzed by western blotting ( a ). Cells were treated with 10 μ M Zea in the presence of LY294002 or U0126 for 24 h, and the protein levels of HO-1 and NQO-1 were analyzed ( b : western blot image; c : statistical analysis of HO-1 expression; d : statistical analysis of NQO-1 expression), GSH levels were also determined ( e ). Following co-treatment with Zea, LY294002, and U0126 for 24 h, cells were challenged with t -BHP for an additional 6 h, after which cell viability ( f ) and the mitochondrial membrane potential ( g ) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (* P
Figure Legend Snippet: Zea activates the Nrf2/Keap1 pathway through PI3/Akt activation. ARPE-19 cells were treated with 10 μ M Zea for the indicated time, and kinase activation was analyzed by western blotting ( a ). Cells were treated with 10 μ M Zea in the presence of LY294002 or U0126 for 24 h, and the protein levels of HO-1 and NQO-1 were analyzed ( b : western blot image; c : statistical analysis of HO-1 expression; d : statistical analysis of NQO-1 expression), GSH levels were also determined ( e ). Following co-treatment with Zea, LY294002, and U0126 for 24 h, cells were challenged with t -BHP for an additional 6 h, after which cell viability ( f ) and the mitochondrial membrane potential ( g ) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (* P

Techniques Used: Activation Assay, Western Blot, Expressing

28) Product Images from "Imidazoline I2 receptor inhibitor idazoxan regulates the progression of hepatic fibrosis via Akt-Nrf2-Smad2/3 signaling pathway"

Article Title: Imidazoline I2 receptor inhibitor idazoxan regulates the progression of hepatic fibrosis via Akt-Nrf2-Smad2/3 signaling pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.15472

IDA activates Nrf2 signaling in CCl4-treated mice and TGF-β-treated LX2 cells ( A ) The expressions of HO-1, NQO-1 and Nrf2 in liver were determined by western blotting (Two randomly samples in each group were presented). ( B – C ) LX2 cells were pretreated with series doses of IDA (25 uM, 50 uM or 100 uM) for 1 h and then treated with or without TGF-β (5 ng/ml) for 1 h. The expressions of HO-1 and NQO-1 were assayed by western blotting. The expressions of Nrf2 and keap1 in total lysate and nucleus were measured by western blotting. ( D ) LX2 cells were treated with PBS or IDA (100 uM) for 1 h. The nuclear translocations of Nrf2 were determined by immunofluorescence assay. ( E ) LX2 cells were pretreated with series doses of IDA (25 uM, 50 uM or 100 uM) for 1 h and then treated with or without TGF-β (5 ng/ml) for 1 h. The DNA binding activity of Nrf2 was measured by EMSA.
Figure Legend Snippet: IDA activates Nrf2 signaling in CCl4-treated mice and TGF-β-treated LX2 cells ( A ) The expressions of HO-1, NQO-1 and Nrf2 in liver were determined by western blotting (Two randomly samples in each group were presented). ( B – C ) LX2 cells were pretreated with series doses of IDA (25 uM, 50 uM or 100 uM) for 1 h and then treated with or without TGF-β (5 ng/ml) for 1 h. The expressions of HO-1 and NQO-1 were assayed by western blotting. The expressions of Nrf2 and keap1 in total lysate and nucleus were measured by western blotting. ( D ) LX2 cells were treated with PBS or IDA (100 uM) for 1 h. The nuclear translocations of Nrf2 were determined by immunofluorescence assay. ( E ) LX2 cells were pretreated with series doses of IDA (25 uM, 50 uM or 100 uM) for 1 h and then treated with or without TGF-β (5 ng/ml) for 1 h. The DNA binding activity of Nrf2 was measured by EMSA.

Techniques Used: Mouse Assay, Western Blot, Immunofluorescence, Binding Assay, Activity Assay

29) Product Images from "Gankyrin has an antioxidative role through the feedback regulation of Nrf2 in hepatocellular carcinoma"

Article Title: Gankyrin has an antioxidative role through the feedback regulation of Nrf2 in hepatocellular carcinoma

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20151208

Gankyrin influenced oxidative stress–induced mitochondrial dysfunction and cell death in HCC cells. (A) Fluorescence microscopy revealed the MitoSOX levels in SMMC7721-con, SMMC7721-ovGank, MHCCLM3con, and MHCCLM3siGank cells. Cells were stained with MitoSOX Red mitochondrial superoxide indicator and the fluorescence value of MitoSOX was quantified. The data represent the mean ± SEM of three independent experiments. Bar, 100 µm. (B) Representative images of the mitochondria ultrastructure were taken by electron microscopy in SMMC7721-con, SMMC7721-siGank, MHCCLM3con, and MHCCLM3siGank cells. Representative results from three experiments are shown. Bars: 1 µm (SMMC7721); 2 µm (MHCCLM3). (C and D) Mitochondrial O 2 consumption assays in MHCCLM3-con, MHCCLM3-siGank cells, SMMC7721-con and SMMC7721-ovGank cells. Cells were treated with PBS or 0.5 mM H 2 O 2 for 5 h. Each data point represents the mean ± SEM of three wells. (E) The overexpression of gankyrin helped SMMC7721 cells gain resistance against oxidative stress–induced death. Cells were incubated with PBS (top), 0.5 mM H 2 O 2 (middle), and 1 mM H 2 O 2 (bottom) for 5 h, and cell death was then observed. Representative results from three experiments are shown. Bar, 100 µm. (F) Suppression of gankyrin-sensitized MHCCLM3 cells to ROS-mediated cell death. MHCCLM3-con and MHCCLM3-siGank cells were treated with H 2 O 2 for 5 h and stained with PI for 20 min, and the number of PI-positive cells was then observed and quantified. The data represent the mean ± SEM of three experiments. Bar, 100 µm. (G) Cleaved PARP was examined in HCC cells with different gankyrin levels upon H 2 O 2 stimulation by Western blotting analysis. Representative results from 3 experiments are shown. (H) NAC treatment attenuated the cell death resulting from gankyrin knockdown. MHCCLM3 cells were transfected with control or gankyrin-siRNA, treated with 100 nM NAC for 48 h, and stained with PI for 15 min. Flow cytometry analysis was performed to evaluate cell apoptosis. Representative results from three experiments are shown. **, P
Figure Legend Snippet: Gankyrin influenced oxidative stress–induced mitochondrial dysfunction and cell death in HCC cells. (A) Fluorescence microscopy revealed the MitoSOX levels in SMMC7721-con, SMMC7721-ovGank, MHCCLM3con, and MHCCLM3siGank cells. Cells were stained with MitoSOX Red mitochondrial superoxide indicator and the fluorescence value of MitoSOX was quantified. The data represent the mean ± SEM of three independent experiments. Bar, 100 µm. (B) Representative images of the mitochondria ultrastructure were taken by electron microscopy in SMMC7721-con, SMMC7721-siGank, MHCCLM3con, and MHCCLM3siGank cells. Representative results from three experiments are shown. Bars: 1 µm (SMMC7721); 2 µm (MHCCLM3). (C and D) Mitochondrial O 2 consumption assays in MHCCLM3-con, MHCCLM3-siGank cells, SMMC7721-con and SMMC7721-ovGank cells. Cells were treated with PBS or 0.5 mM H 2 O 2 for 5 h. Each data point represents the mean ± SEM of three wells. (E) The overexpression of gankyrin helped SMMC7721 cells gain resistance against oxidative stress–induced death. Cells were incubated with PBS (top), 0.5 mM H 2 O 2 (middle), and 1 mM H 2 O 2 (bottom) for 5 h, and cell death was then observed. Representative results from three experiments are shown. Bar, 100 µm. (F) Suppression of gankyrin-sensitized MHCCLM3 cells to ROS-mediated cell death. MHCCLM3-con and MHCCLM3-siGank cells were treated with H 2 O 2 for 5 h and stained with PI for 20 min, and the number of PI-positive cells was then observed and quantified. The data represent the mean ± SEM of three experiments. Bar, 100 µm. (G) Cleaved PARP was examined in HCC cells with different gankyrin levels upon H 2 O 2 stimulation by Western blotting analysis. Representative results from 3 experiments are shown. (H) NAC treatment attenuated the cell death resulting from gankyrin knockdown. MHCCLM3 cells were transfected with control or gankyrin-siRNA, treated with 100 nM NAC for 48 h, and stained with PI for 15 min. Flow cytometry analysis was performed to evaluate cell apoptosis. Representative results from three experiments are shown. **, P

Techniques Used: Fluorescence, Microscopy, Staining, Electron Microscopy, Over Expression, Incubation, Western Blot, Transfection, Flow Cytometry, Cytometry

Gankyrin and Nrf2 overexpression in HCCs predicts a poor prognosis. (A and B) Livers of Wistar rats euthanized at week 6, 8, 14, or 20 wk after DEN administration were collected and subjected to immunohistochemistry with anti-gankyrin (A) and anti-Nrf2 (B) antibodies. Bars:1 mm (top), 300 µm (bottom); n = 5 for each time point. (C) Gankyrin expression positively correlated with Nrf2 target genes in HCC specimens. The levels of gankyrin and Nrf2 target genes mRNA were detected by qRT-PCR, and the correlations between the mRNA levels of gankyrin and various antioxidative enzymes were evaluated; n = 86. (D) Immunohistochemical staining of gankyrin and Nrf2 protein levels in HCC TMA sections. Representative staining of gankyrin and Nrf2 is shown. Bar, 200 µm. (E) Graphical representation of the distribution of patients according to the staining intensities of gankyrin and Nrf2 in HCCs. (F) Kaplan-Meier curves for time to recurrence and overall survival of patients among the different groups shown in E.
Figure Legend Snippet: Gankyrin and Nrf2 overexpression in HCCs predicts a poor prognosis. (A and B) Livers of Wistar rats euthanized at week 6, 8, 14, or 20 wk after DEN administration were collected and subjected to immunohistochemistry with anti-gankyrin (A) and anti-Nrf2 (B) antibodies. Bars:1 mm (top), 300 µm (bottom); n = 5 for each time point. (C) Gankyrin expression positively correlated with Nrf2 target genes in HCC specimens. The levels of gankyrin and Nrf2 target genes mRNA were detected by qRT-PCR, and the correlations between the mRNA levels of gankyrin and various antioxidative enzymes were evaluated; n = 86. (D) Immunohistochemical staining of gankyrin and Nrf2 protein levels in HCC TMA sections. Representative staining of gankyrin and Nrf2 is shown. Bar, 200 µm. (E) Graphical representation of the distribution of patients according to the staining intensities of gankyrin and Nrf2 in HCCs. (F) Kaplan-Meier curves for time to recurrence and overall survival of patients among the different groups shown in E.

Techniques Used: Over Expression, Immunohistochemistry, Expressing, Quantitative RT-PCR, Staining

Nrf2 promotes gankyrin transcription. (A) Schematic representation of gankyrin promoters in humans ( Homo species , hGank), pigs ( Sus scrofa , sGank), mice ( Mus musculus , mGank), rats ( Rattus norvegicus , rGank), and chimpanzees ( Pan troglodytes , pGank). Different symbols represent Nrf2 binding sites (ARE) in different species. TSS, transcriptional start site. (B) ARE regions and adjacent sequences in the gankyrin promoter. (C) SMMC7721 and MHCC-LM3 cells were fixed and sheered; cross-linked chromatin was prepared as described in the Materials and methods. The chromatin was precipitated using control (IgG) or Nrf2-specific antibodies (Nrf2). PCR analysis was performed using primers for ARE1, ARE2, and ARE3. The data shown are representative of three independent experiments. (D) Oxidative stress increases the binding of Nrf2 to the AREs of the gankyrin promoter. SMMC7721 and MHCCLM3 cells were treated with PBS or 0.5 mM H 2 O 2 for 5 h, and chromatin immunoprecipitation was performed using Nrf2-specific antibodies. DNA isolated from the precipitated materials was analyzed using qPCR with the indicated primers. The ARE-specific signals from Nrf2-precipitated DNA were normalized to those from IgG-precipitated DNA. The data shown are means ± SEM of triplicate wells. (E and F) qRT-PCR analysis was performed for gankyrin and target genes of Nrf2 in PLC/RPF/5-con and PLC/RPF/5-siNrf2 or SMMC7721-con and SMMC7721-siNrf2 cells. Data represent the mean ± SEM of triplicates from an experiment that was repeated a total of three times with similar results. *, P
Figure Legend Snippet: Nrf2 promotes gankyrin transcription. (A) Schematic representation of gankyrin promoters in humans ( Homo species , hGank), pigs ( Sus scrofa , sGank), mice ( Mus musculus , mGank), rats ( Rattus norvegicus , rGank), and chimpanzees ( Pan troglodytes , pGank). Different symbols represent Nrf2 binding sites (ARE) in different species. TSS, transcriptional start site. (B) ARE regions and adjacent sequences in the gankyrin promoter. (C) SMMC7721 and MHCC-LM3 cells were fixed and sheered; cross-linked chromatin was prepared as described in the Materials and methods. The chromatin was precipitated using control (IgG) or Nrf2-specific antibodies (Nrf2). PCR analysis was performed using primers for ARE1, ARE2, and ARE3. The data shown are representative of three independent experiments. (D) Oxidative stress increases the binding of Nrf2 to the AREs of the gankyrin promoter. SMMC7721 and MHCCLM3 cells were treated with PBS or 0.5 mM H 2 O 2 for 5 h, and chromatin immunoprecipitation was performed using Nrf2-specific antibodies. DNA isolated from the precipitated materials was analyzed using qPCR with the indicated primers. The ARE-specific signals from Nrf2-precipitated DNA were normalized to those from IgG-precipitated DNA. The data shown are means ± SEM of triplicate wells. (E and F) qRT-PCR analysis was performed for gankyrin and target genes of Nrf2 in PLC/RPF/5-con and PLC/RPF/5-siNrf2 or SMMC7721-con and SMMC7721-siNrf2 cells. Data represent the mean ± SEM of triplicates from an experiment that was repeated a total of three times with similar results. *, P

Techniques Used: Mouse Assay, Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Planar Chromatography

Gankyrin increases the expression of antioxidative enzymes. (A and B) qRT-PCR analysis was performed for SOD1, SOD2, ANT, CAT, Gpx, GCLC, GCLM, HO-1, NQO1, and AKR family members and gankyrin in MHCCLM3 and SMMC7721 control and gankyrin knockdown cells. The data are the mean ± the SEM of three independent experiments. (C) qRT-PCR analysis was performed to detect SOD1, SOD2, ANT, and Gpx expression in SMMC7721 cells that were transiently transfected with gankyrin-pcDNA3.1A vectors with or without H 2 O 2 stimulation. The data are expressed as the mean ± SEM of three independent experiments. (D) Western blot analysis showed the levels of NQO1, HO-1, and GCLM protein in SMMC7721-con, SMMC7721-siGank, MHCCLM3-con, and MHCCLM3-siGank cells. The data shown are representative of three independent experiments. (E) Western blotting analysis showed the levels of AKR1B10 and AKR1C3 protein in gankyrin-knockdown or overexpressing SMMC7721 cells. The data shown are representative of three independent experiments. (F) Gankyrin influenced ARE luciferase reporter activity in HCC cells. MHCCLM3 and SMMC7721 cells with different gankyrin levels were transiently transfected with an ARE luciferase reporter vector or the control plasmid pRL-TK for 48 h. The cells were harvested and gankyrin reporter activities were detected. The results are means ± SEM. n = 3. *, P
Figure Legend Snippet: Gankyrin increases the expression of antioxidative enzymes. (A and B) qRT-PCR analysis was performed for SOD1, SOD2, ANT, CAT, Gpx, GCLC, GCLM, HO-1, NQO1, and AKR family members and gankyrin in MHCCLM3 and SMMC7721 control and gankyrin knockdown cells. The data are the mean ± the SEM of three independent experiments. (C) qRT-PCR analysis was performed to detect SOD1, SOD2, ANT, and Gpx expression in SMMC7721 cells that were transiently transfected with gankyrin-pcDNA3.1A vectors with or without H 2 O 2 stimulation. The data are expressed as the mean ± SEM of three independent experiments. (D) Western blot analysis showed the levels of NQO1, HO-1, and GCLM protein in SMMC7721-con, SMMC7721-siGank, MHCCLM3-con, and MHCCLM3-siGank cells. The data shown are representative of three independent experiments. (E) Western blotting analysis showed the levels of AKR1B10 and AKR1C3 protein in gankyrin-knockdown or overexpressing SMMC7721 cells. The data shown are representative of three independent experiments. (F) Gankyrin influenced ARE luciferase reporter activity in HCC cells. MHCCLM3 and SMMC7721 cells with different gankyrin levels were transiently transfected with an ARE luciferase reporter vector or the control plasmid pRL-TK for 48 h. The cells were harvested and gankyrin reporter activities were detected. The results are means ± SEM. n = 3. *, P

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Luciferase, Activity Assay, Plasmid Preparation

Gankyrin binds to the Kelch domain of Keap1. (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.
Figure Legend Snippet: Gankyrin binds to the Kelch domain of Keap1. (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.

Techniques Used: Binding Assay, Immunoprecipitation, Confocal Microscopy, Western Blot, Transfection, Negative Control, Co-Immunoprecipitation Assay

Gankyrin expression is increased under oxidative stress and participated in elimination of ROS . (A) qRT-PCR analysis of gankyrin expression in SMMC7721, PLC/PRF/5, and MHCCLM3 cells. The data are the mean ± SEM of three independent experiments. (B) Western blot analysis of gankyrin expression at different time points or after different concentrations of H 2 O 2 treatment in MHCCLM3 cells. (C) Western blot of gankyrin expression in MHCC-LM3 cells treated with 100 nM NAC for 24 to 72 h; the protein levels were quantified relative to the loading control. (D) ROS levels were detected in MHCCLM3 gankyrin knockdown and control cells. Cells were treated with PBS or 0.5 mM of H 2 O 2 for 5 h, and the cells were then incubated with CM-H2DCFDA for 30 min. (E) Flow cytometry analysis to detect ROS levels in gankyrin-overexpressing and control SMMC7721 cells. (F) Fluorescence microscopy to detect ROS levels in SMMC7721 cells transiently transfected with gankyrin overexpressing plasmid. Bar, 100 µm. (G) Gankyrin regulated the total antioxidant capacity of HCC cells. SMMC7721 and MHCCLM3 cells with different gankyrin levels were treated with 0.5 mM H 2 O 2 or PBS for 5 h, and the total antioxidant capacity was then measured with a T-AOC Assay kit. The results are the means ± SEM of three independent experiments. Data in B–F are representative of at least three experiments with similar results.*, P
Figure Legend Snippet: Gankyrin expression is increased under oxidative stress and participated in elimination of ROS . (A) qRT-PCR analysis of gankyrin expression in SMMC7721, PLC/PRF/5, and MHCCLM3 cells. The data are the mean ± SEM of three independent experiments. (B) Western blot analysis of gankyrin expression at different time points or after different concentrations of H 2 O 2 treatment in MHCCLM3 cells. (C) Western blot of gankyrin expression in MHCC-LM3 cells treated with 100 nM NAC for 24 to 72 h; the protein levels were quantified relative to the loading control. (D) ROS levels were detected in MHCCLM3 gankyrin knockdown and control cells. Cells were treated with PBS or 0.5 mM of H 2 O 2 for 5 h, and the cells were then incubated with CM-H2DCFDA for 30 min. (E) Flow cytometry analysis to detect ROS levels in gankyrin-overexpressing and control SMMC7721 cells. (F) Fluorescence microscopy to detect ROS levels in SMMC7721 cells transiently transfected with gankyrin overexpressing plasmid. Bar, 100 µm. (G) Gankyrin regulated the total antioxidant capacity of HCC cells. SMMC7721 and MHCCLM3 cells with different gankyrin levels were treated with 0.5 mM H 2 O 2 or PBS for 5 h, and the total antioxidant capacity was then measured with a T-AOC Assay kit. The results are the means ± SEM of three independent experiments. Data in B–F are representative of at least three experiments with similar results.*, P

Techniques Used: Expressing, Quantitative RT-PCR, Planar Chromatography, Western Blot, Incubation, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Transfection, Plasmid Preparation

Nrf2 and gankyrin cooperatively provide HCC cells with increased antioxidative stress capacity. (A) MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, and MHCCLM3-siGankovNrf2 or SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 cells were treated with 0.5 mM H 2 O 2 for 5 h and stained with PI for 15 min. Flow cytometry assays were performed to evaluate the level of apoptosis in HCC cells. Representative results from three experiments are shown. (B) Fluorescence microscopy showed the levels of cell death in MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, MHCCLM3-siGankovNrf2 (top), SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 (bottom) cells. Cells were treated with 0.5 mM H 2 O 2 for 5 h and stained with PI for 15 min. Representative results from three experiments are shown. Bar, 100 µM. (C) Levels of cleaved PARP were evaluated in MHCCLM3-con, MHCCLM3-siGank, and MHCCLM3-siGankovNrf2 cells (top) or SMMC7721-con, SMMC7721-ovGank, and SMMC7721-ovGanksiNrf2 cells treated with 0.5 mM H 2 O 2 for the indicated time. Representative results from three experiments are shown. (D) Quantification of the mitoSOX levels in MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, MHCCLM3-siGankovNrf2, SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 cells. The data represent the mean ± SEM of triplicates from an experiment that was repeated a total of three times with similar results. (E) Flow cytometry analyses were performed on MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, MHCCLM3-siGankovNrf2, and SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 cells to detect the levels of ROS. Representative results from three experiments are shown. (F) HCC cells were transiently transfected with an ARE luciferase reporter vector or the control plasmid pRL-TK for 48 h. The cells were then harvested, and the reporter activities were detected. The data represent the mean ± SEM of triplicates from an experiment that was repeated a total of three times with similar results. (G and H) Mitochondrial O 2 consumption assays in MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-siGankovNrf2, and SMMC7721-con, SMMC7721-ovGank, and SMMC7721-ovGanksiNrf2 cells. Cells were treated with PBS or 0.5 mM H 2 O 2 for 5 h, and O 2 consumption was then examined. Each data point represents the mean ± SEM of triplicates from an experiment that was repeated three times with similar results. (I and J) Tumors were excised from nude mice 25 d after subcutaneous inoculation with SMMC7721-con, SMMC7721-ovGank, or SMMC7721-ovGanksiNrf2 cells. Tumor size was measured once every 3 d, and the overall tumor volume was calculated. The data represent the mean ± SEM. n = 5. *, P
Figure Legend Snippet: Nrf2 and gankyrin cooperatively provide HCC cells with increased antioxidative stress capacity. (A) MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, and MHCCLM3-siGankovNrf2 or SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 cells were treated with 0.5 mM H 2 O 2 for 5 h and stained with PI for 15 min. Flow cytometry assays were performed to evaluate the level of apoptosis in HCC cells. Representative results from three experiments are shown. (B) Fluorescence microscopy showed the levels of cell death in MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, MHCCLM3-siGankovNrf2 (top), SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 (bottom) cells. Cells were treated with 0.5 mM H 2 O 2 for 5 h and stained with PI for 15 min. Representative results from three experiments are shown. Bar, 100 µM. (C) Levels of cleaved PARP were evaluated in MHCCLM3-con, MHCCLM3-siGank, and MHCCLM3-siGankovNrf2 cells (top) or SMMC7721-con, SMMC7721-ovGank, and SMMC7721-ovGanksiNrf2 cells treated with 0.5 mM H 2 O 2 for the indicated time. Representative results from three experiments are shown. (D) Quantification of the mitoSOX levels in MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, MHCCLM3-siGankovNrf2, SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 cells. The data represent the mean ± SEM of triplicates from an experiment that was repeated a total of three times with similar results. (E) Flow cytometry analyses were performed on MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-ovNrf2, MHCCLM3-siGankovNrf2, and SMMC7721-con, SMMC7721-ovGank, SMMC7721-siNrf2, and SMMC7721-ovGanksiNrf2 cells to detect the levels of ROS. Representative results from three experiments are shown. (F) HCC cells were transiently transfected with an ARE luciferase reporter vector or the control plasmid pRL-TK for 48 h. The cells were then harvested, and the reporter activities were detected. The data represent the mean ± SEM of triplicates from an experiment that was repeated a total of three times with similar results. (G and H) Mitochondrial O 2 consumption assays in MHCCLM3-con, MHCCLM3-siGank, MHCCLM3-siGankovNrf2, and SMMC7721-con, SMMC7721-ovGank, and SMMC7721-ovGanksiNrf2 cells. Cells were treated with PBS or 0.5 mM H 2 O 2 for 5 h, and O 2 consumption was then examined. Each data point represents the mean ± SEM of triplicates from an experiment that was repeated three times with similar results. (I and J) Tumors were excised from nude mice 25 d after subcutaneous inoculation with SMMC7721-con, SMMC7721-ovGank, or SMMC7721-ovGanksiNrf2 cells. Tumor size was measured once every 3 d, and the overall tumor volume was calculated. The data represent the mean ± SEM. n = 5. *, P

Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Transfection, Luciferase, Plasmid Preparation, Mouse Assay

30) Product Images from "EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation"

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation

Journal: Autophagy

doi: 10.1080/15548627.2018.1536530

SQSTM1 accumulation activates the SQSTM1-KEAP1-NFE2L2 axis that reduces ROS production in EBV-infected monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) KEAP1 and NFE2L2 expression by western blot, (b) NFE2L2 localization by IFA and (c) CAT and GSR expression by western blot. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of KEAP1:ACTB, NFE2L2:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. SQSTM1 staining is shown in red; bars: 10 mm. Differentiating monocytes exposed to EBV were silenced for si SQSTM1 with specific siRNA or scrambled siRNA and cultured for 5 days with CSF2 and IL4 before analysing (d) NFE2L2 localization by IFA and (e) SQSTM1, CAT and GSR expression by western blot. NFE2L2 staining is shown in red; bars: 10 mm. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. * P value
Figure Legend Snippet: SQSTM1 accumulation activates the SQSTM1-KEAP1-NFE2L2 axis that reduces ROS production in EBV-infected monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) KEAP1 and NFE2L2 expression by western blot, (b) NFE2L2 localization by IFA and (c) CAT and GSR expression by western blot. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of KEAP1:ACTB, NFE2L2:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. SQSTM1 staining is shown in red; bars: 10 mm. Differentiating monocytes exposed to EBV were silenced for si SQSTM1 with specific siRNA or scrambled siRNA and cultured for 5 days with CSF2 and IL4 before analysing (d) NFE2L2 localization by IFA and (e) SQSTM1, CAT and GSR expression by western blot. NFE2L2 staining is shown in red; bars: 10 mm. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. * P value

Techniques Used: Infection, Cell Culture, Expressing, Western Blot, Immunofluorescence, Staining

31) Product Images from "The synthetic triterpenoid RTA dh404 (CDDO-dhTFEA) restores endothelial function impaired by reduced Nrf2 activity in chronic kidney disease"

Article Title: The synthetic triterpenoid RTA dh404 (CDDO-dhTFEA) restores endothelial function impaired by reduced Nrf2 activity in chronic kidney disease

Journal: Redox Biology

doi: 10.1016/j.redox.2013.10.007

Effect of RTA dh404 on Nrf2, Nrf2 target, and Keap1 protein expression in aorta from CKD rats . Representative Western blots and group data are presented, depicting protein abundance of Nrf2, Nrf2 downstream gene products: superoxide dismutase 2 (Sod2) and heme oxygenase-1 (Ho-1), as well as Keap1 in the aortas of sham-operated control ( n =6) and 5/6 nephrectomized rats [chronic renal failure (CKD)] treated with vehicle (CKD; n =9) or RTA dh404 (CKD+RTA dh404; n =9). Histone H1 served as the loading control for Nrf2, whereas GAPDH served as the loading control for Sod2, Ho-1, and Keap1. Asterisks indicate a statistically significant difference from sham control ( ⁎ p
Figure Legend Snippet: Effect of RTA dh404 on Nrf2, Nrf2 target, and Keap1 protein expression in aorta from CKD rats . Representative Western blots and group data are presented, depicting protein abundance of Nrf2, Nrf2 downstream gene products: superoxide dismutase 2 (Sod2) and heme oxygenase-1 (Ho-1), as well as Keap1 in the aortas of sham-operated control ( n =6) and 5/6 nephrectomized rats [chronic renal failure (CKD)] treated with vehicle (CKD; n =9) or RTA dh404 (CKD+RTA dh404; n =9). Histone H1 served as the loading control for Nrf2, whereas GAPDH served as the loading control for Sod2, Ho-1, and Keap1. Asterisks indicate a statistically significant difference from sham control ( ⁎ p

Techniques Used: Expressing, Western Blot

32) Product Images from "Huang-Lian-Jie-Du Decoction Ameliorates Acute Ulcerative Colitis in Mice via Regulating NF-κB and Nrf2 Signaling Pathways and Enhancing Intestinal Barrier Function"

Article Title: Huang-Lian-Jie-Du Decoction Ameliorates Acute Ulcerative Colitis in Mice via Regulating NF-κB and Nrf2 Signaling Pathways and Enhancing Intestinal Barrier Function

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.01354

The regulation effect of HLJDD on Nrf2 signaling pathway. HLJDD therapy up-regulates the expression of key proteins Nrf2 (A) and Keap1 (B) involved in the Nrf2 signaling pathway in colon; (C) Immunofluorescence analysis of Nrf2 (green) in colon mucosa (600 × magnification). DAPI was used for nuclear staining (blue). Data are expressed as the mean ± SD, n = 5 per group. ### p
Figure Legend Snippet: The regulation effect of HLJDD on Nrf2 signaling pathway. HLJDD therapy up-regulates the expression of key proteins Nrf2 (A) and Keap1 (B) involved in the Nrf2 signaling pathway in colon; (C) Immunofluorescence analysis of Nrf2 (green) in colon mucosa (600 × magnification). DAPI was used for nuclear staining (blue). Data are expressed as the mean ± SD, n = 5 per group. ### p

Techniques Used: Expressing, Immunofluorescence, Staining

33) Product Images from "Keap1 degradation by autophagy for the maintenance of redox homeostasis"

Article Title: Keap1 degradation by autophagy for the maintenance of redox homeostasis

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1121572109

Accumulation of p62 in Atg7 :: Keap1-Alb :: Nrf2 −/− liver. Immunoblot analysis of whole-cell extracts (Whole Cell) and nuclear extracts (Nucleus) of liver ( A ) and relative mRNA expression measured by quantitative real-time PCR ( B ) in livers from various genotypes of mice. αTubulin and Lamin B were used as loading controls. Lanes 1 Atg7 flox/flox (Control), 2 Atg7 flox/flox - Alb , 3 Atg7 flox/flox :: Keap1 flox/flox - Alb , 4 Atg7 flox/flox :: Keap1 flox/flox - Alb :: p62 −/− , and 5 Atg7 flox/flox :: Keap1 flox/flox - Alb :: Nrf2 −/− . Data are the means ± SD. * P
Figure Legend Snippet: Accumulation of p62 in Atg7 :: Keap1-Alb :: Nrf2 −/− liver. Immunoblot analysis of whole-cell extracts (Whole Cell) and nuclear extracts (Nucleus) of liver ( A ) and relative mRNA expression measured by quantitative real-time PCR ( B ) in livers from various genotypes of mice. αTubulin and Lamin B were used as loading controls. Lanes 1 Atg7 flox/flox (Control), 2 Atg7 flox/flox - Alb , 3 Atg7 flox/flox :: Keap1 flox/flox - Alb , 4 Atg7 flox/flox :: Keap1 flox/flox - Alb :: p62 −/− , and 5 Atg7 flox/flox :: Keap1 flox/flox - Alb :: Nrf2 −/− . Data are the means ± SD. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

Increase of the Keap1 protein level in Atg7-Alb and p62 −/− mice. Protein and mRNA expression levels of Keap1, Nrf2, and p62 in Nrf2 −/− , Atg7-Alb , Atg7-Alb :: Nrf2 −/− , and Keap1-Alb mice ( A and B ) and p62 −/− mice ( C and D ). Immunoblot analyses were carried out using whole-cell extracts (Whole Cell) and nuclear extracts (Nucleus) of liver ( A and C ). αTubulin and Lamin B were used as loading controls. Atg7-Alb was used as a positive control in C . Data are the means ± SD of three mice. * P
Figure Legend Snippet: Increase of the Keap1 protein level in Atg7-Alb and p62 −/− mice. Protein and mRNA expression levels of Keap1, Nrf2, and p62 in Nrf2 −/− , Atg7-Alb , Atg7-Alb :: Nrf2 −/− , and Keap1-Alb mice ( A and B ) and p62 −/− mice ( C and D ). Immunoblot analyses were carried out using whole-cell extracts (Whole Cell) and nuclear extracts (Nucleus) of liver ( A and C ). αTubulin and Lamin B were used as loading controls. Atg7-Alb was used as a positive control in C . Data are the means ± SD of three mice. * P

Techniques Used: Mouse Assay, Expressing, Positive Control

34) Product Images from "EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation"

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation

Journal: Autophagy

doi: 10.1080/15548627.2018.1536530

EBV reduces mitochondrial biogenesis in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) CYCS and (b) for NRF1 and TFAM expression by western blot. ACTB was used as a loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of CYCS:ACTB, NRF1:ACTB and TFAM:ACTB of 3 different experiments. * P value
Figure Legend Snippet: EBV reduces mitochondrial biogenesis in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) CYCS and (b) for NRF1 and TFAM expression by western blot. ACTB was used as a loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of CYCS:ACTB, NRF1:ACTB and TFAM:ACTB of 3 different experiments. * P value

Techniques Used: Cell Culture, Expressing, Western Blot

35) Product Images from "Protective action of glutamine in rats with severe acute liver failure"

Article Title: Protective action of glutamine in rats with severe acute liver failure

Journal: World Journal of Hepatology

doi: 10.4254/wjh.v11.i3.273

Effect of glutamine in experimental severe acute liver failure. Western blot analysis of protein expression of (A) TLR4 and (B) NFκB. Values expressed as mean ± SE. b P
Figure Legend Snippet: Effect of glutamine in experimental severe acute liver failure. Western blot analysis of protein expression of (A) TLR4 and (B) NFκB. Values expressed as mean ± SE. b P

Techniques Used: Western Blot, Expressing

36) Product Images from "Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor"

Article Title: Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00037-19

Design and validation of the ARE-BSD-PEST reporter. (A) ARE-BSD-PEST consists of a transcriptional pause site (TPS) followed by a synthetic sequence consisting of a triple antioxidant response sequence (4× ARE), a minimal promoter, an open reading frame for a blasticidin resistance gene with a PEST degron sequence (BSD-PEST), and a terminator; the BSD-PEST transcription is under the control of the NRF2 transcription factor. (B) Western blot showing effective KEAP1 knockout. The Western blot shows activation of NRF2 as well as NRF2 transcriptional targets NQO1 and FTL. β-Actin (ACTB) was used as a loading control. (C) HK2-BSD cells harboring KEAP1 knockout (HK2-BSD-sgKEAP1) are more resistant to blasticidin than control cells.
Figure Legend Snippet: Design and validation of the ARE-BSD-PEST reporter. (A) ARE-BSD-PEST consists of a transcriptional pause site (TPS) followed by a synthetic sequence consisting of a triple antioxidant response sequence (4× ARE), a minimal promoter, an open reading frame for a blasticidin resistance gene with a PEST degron sequence (BSD-PEST), and a terminator; the BSD-PEST transcription is under the control of the NRF2 transcription factor. (B) Western blot showing effective KEAP1 knockout. The Western blot shows activation of NRF2 as well as NRF2 transcriptional targets NQO1 and FTL. β-Actin (ACTB) was used as a loading control. (C) HK2-BSD cells harboring KEAP1 knockout (HK2-BSD-sgKEAP1) are more resistant to blasticidin than control cells.

Techniques Used: Sequencing, Western Blot, Knock-Out, Activation Assay

37) Product Images from "Astragaloside IV Attenuates Acetaminophen-Induced Liver Injuries in Mice by Activating the Nrf2 Signaling Pathway"

Article Title: Astragaloside IV Attenuates Acetaminophen-Induced Liver Injuries in Mice by Activating the Nrf2 Signaling Pathway

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23082032

AS-IV activated Nrf2 signaling pathway ( n = 3). Western blotting analysis of Nrf2, Keap1, HO-1, and NQO1.
Figure Legend Snippet: AS-IV activated Nrf2 signaling pathway ( n = 3). Western blotting analysis of Nrf2, Keap1, HO-1, and NQO1.

Techniques Used: Western Blot

38) Product Images from "Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells"

Article Title: Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22060969

Role of Nrf2 in DHA-induced expression of HO-1 and NQO1. ( A ) MCF-10A cells were transfected with siRNA control, siRNA Nrf2, or siRNA Nrf2 plus Nrf2 full-sequence vector for 12 h and exposed to DHA (25 μM) for another 9 h. Whole cell lysates were subjected to Western blot analysis. ( B ) Nrf2-WT or Nrf2-null MEF cells were incubated with 25 μM of DHA for 12 h, and the expression of Nrf2, HO-1, and NQO1 was measured by Western blot analysis. ( C ) MCF-10A cells were treated with DHA (25 μM) for 9 h and harvested to determine the ARE binding activity by the ChIP assay. Chromatin immunoprecipitated DNA was analyzed by RT-PCR with primers for distal E2 (−9.0 kb region) and E1 (−4.0 kb region) AREs as well as non-specific region (after −9.0 kb) of the HO-1 promoter.
Figure Legend Snippet: Role of Nrf2 in DHA-induced expression of HO-1 and NQO1. ( A ) MCF-10A cells were transfected with siRNA control, siRNA Nrf2, or siRNA Nrf2 plus Nrf2 full-sequence vector for 12 h and exposed to DHA (25 μM) for another 9 h. Whole cell lysates were subjected to Western blot analysis. ( B ) Nrf2-WT or Nrf2-null MEF cells were incubated with 25 μM of DHA for 12 h, and the expression of Nrf2, HO-1, and NQO1 was measured by Western blot analysis. ( C ) MCF-10A cells were treated with DHA (25 μM) for 9 h and harvested to determine the ARE binding activity by the ChIP assay. Chromatin immunoprecipitated DNA was analyzed by RT-PCR with primers for distal E2 (−9.0 kb region) and E1 (−4.0 kb region) AREs as well as non-specific region (after −9.0 kb) of the HO-1 promoter.

Techniques Used: Expressing, Transfection, Sequencing, Plasmid Preparation, Western Blot, Incubation, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Role of ROS in DHA-induced Nrf2 activation mediated by PKCδ. ( A ) MCF-10A cells were treated with each indicated drug (EPA and DHA; 25 μM, NAC; 5 mM, H 2 O 2 ; 100 μM) or in proper combination for 3 h and then examined for the intracellular accumulation of ROS under the confocal microscope after DCF-DA fluorescence staining. ( B ) Cells were treated with DHA (25 μM) for the indicated time periods, and the effect of DHA on PKCδ activation was determined by Western blot analysis. ( C ) MCF-10A cells treated with DHA (25 μM) were harvested at the indicated time intervals. The effect of DHA on the phosphorylation of Ser 40 at Nrf2 was assessed by Western blot analysis. Each blot is a representative of three different experiments. Columns, means (n = 3); bars, SD. * p
Figure Legend Snippet: Role of ROS in DHA-induced Nrf2 activation mediated by PKCδ. ( A ) MCF-10A cells were treated with each indicated drug (EPA and DHA; 25 μM, NAC; 5 mM, H 2 O 2 ; 100 μM) or in proper combination for 3 h and then examined for the intracellular accumulation of ROS under the confocal microscope after DCF-DA fluorescence staining. ( B ) Cells were treated with DHA (25 μM) for the indicated time periods, and the effect of DHA on PKCδ activation was determined by Western blot analysis. ( C ) MCF-10A cells treated with DHA (25 μM) were harvested at the indicated time intervals. The effect of DHA on the phosphorylation of Ser 40 at Nrf2 was assessed by Western blot analysis. Each blot is a representative of three different experiments. Columns, means (n = 3); bars, SD. * p

Techniques Used: Activation Assay, Microscopy, Fluorescence, Staining, Western Blot

DHA-induced expression, nuclear translocation and transcriptional activity of Nrf2. ( A ) Total RNA was isolated from cells treated with or without DHA for indicated duration and analyzed by RT-PCR for detecting the level of Nrf2 mRNA; ( B ) MCF-10Acells were exposed to DHA (25 μM) were harvested at the indicated intervals, and the protein levels were assessed by Western blot analysis. ( C ) Nuclear extracts from MCF-10A cells were prepared at the indicated intervals after treatment with DHA (25 μM). ( D ) MCF-10A cells were treated with indicated concentrations of DHA for 9 h and the nuclear translocation of Nrf2 was assessed by Western blot analysis. ( E ) MCF-10A cells were incubated with DHA (25 μM) for 9 h and nuclear localization of Nrf2 was determined by immunocytochemical analysis. ( F ) MCF-10A cells were treated with DHA (25 μM) for 9 h after transfection with either an ARE luciferase construct or a control vector and analyzed for the Nrf2 transcriptional activity as described in Materials and Methods. Columns, means (n = 3); bars, SD. ***, p
Figure Legend Snippet: DHA-induced expression, nuclear translocation and transcriptional activity of Nrf2. ( A ) Total RNA was isolated from cells treated with or without DHA for indicated duration and analyzed by RT-PCR for detecting the level of Nrf2 mRNA; ( B ) MCF-10Acells were exposed to DHA (25 μM) were harvested at the indicated intervals, and the protein levels were assessed by Western blot analysis. ( C ) Nuclear extracts from MCF-10A cells were prepared at the indicated intervals after treatment with DHA (25 μM). ( D ) MCF-10A cells were treated with indicated concentrations of DHA for 9 h and the nuclear translocation of Nrf2 was assessed by Western blot analysis. ( E ) MCF-10A cells were incubated with DHA (25 μM) for 9 h and nuclear localization of Nrf2 was determined by immunocytochemical analysis. ( F ) MCF-10A cells were treated with DHA (25 μM) for 9 h after transfection with either an ARE luciferase construct or a control vector and analyzed for the Nrf2 transcriptional activity as described in Materials and Methods. Columns, means (n = 3); bars, SD. ***, p

Techniques Used: Expressing, Translocation Assay, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation, Transfection, Luciferase, Construct, Plasmid Preparation

39) Product Images from "α-Lipoic Acid Inhibits IL-8 Expression by Activating Nrf2 Signaling in Helicobacter pylori-infected Gastric Epithelial Cells"

Article Title: α-Lipoic Acid Inhibits IL-8 Expression by Activating Nrf2 Signaling in Helicobacter pylori-infected Gastric Epithelial Cells

Journal: Nutrients

doi: 10.3390/nu11102524

Determination of the impact of α-LA on the levels of activated Nrf2, KEAP 1-bound Nrf2, KEAP 1, and HO-1 in AGS cells. ( A ) Western blots of whole-cell extracts (upper panel) or nuclear extracts (bottom panel) with actin serving as the loading control and histone H1 serving as the index for the nuclear extracts. The cells were treated with 5 μM α-LA for the indicated time periods. ( B ) Confocal microscope images of AGS cells treated with 5 μM α-LA for 2 h followed by immunofluorescence staining of the fixed cells. Nrf2 was visualized using fluorescein rhodamin-conjugated anti-rabbit IgG antibody (red) with DAPI counter staining (blue) of the same field. “None” refers to the cells treated with the vehicle for α-LA (0.5 M ethanol) alone. ( C ) Western blots of whole-cell extracts (lower panel) and whole-cell extract-derived immunoprecipitates obtained using the anti-Nrf2 and anti-KEAP 1 antibodies for precipitation (IP) and visualization (WB; western blot analysis) as indicated (upper panel). The cells were treated with 5 μM α-LA for 2 h. Input is used as the control for protein expression. “None” refers to the cells treated with the vehicle for α-LA (0.5 M ethanol) only.
Figure Legend Snippet: Determination of the impact of α-LA on the levels of activated Nrf2, KEAP 1-bound Nrf2, KEAP 1, and HO-1 in AGS cells. ( A ) Western blots of whole-cell extracts (upper panel) or nuclear extracts (bottom panel) with actin serving as the loading control and histone H1 serving as the index for the nuclear extracts. The cells were treated with 5 μM α-LA for the indicated time periods. ( B ) Confocal microscope images of AGS cells treated with 5 μM α-LA for 2 h followed by immunofluorescence staining of the fixed cells. Nrf2 was visualized using fluorescein rhodamin-conjugated anti-rabbit IgG antibody (red) with DAPI counter staining (blue) of the same field. “None” refers to the cells treated with the vehicle for α-LA (0.5 M ethanol) alone. ( C ) Western blots of whole-cell extracts (lower panel) and whole-cell extract-derived immunoprecipitates obtained using the anti-Nrf2 and anti-KEAP 1 antibodies for precipitation (IP) and visualization (WB; western blot analysis) as indicated (upper panel). The cells were treated with 5 μM α-LA for 2 h. Input is used as the control for protein expression. “None” refers to the cells treated with the vehicle for α-LA (0.5 M ethanol) only.

Techniques Used: Western Blot, Microscopy, Immunofluorescence, Staining, Derivative Assay, Expressing

Determination of the impact of the Nrf2 inhibitor trigonelline on IL-8 gene expression and ROS levels in H. pylori -infected AGS cells treated with α-LA. The cells were treated with 5 μM α-LA and 1 μM trigonelline for 8 h, and then infected with H. pylori . ( A ) The amount of IL-8 mRNA determined by real-time PCR analysis and normalized to cellular actin mRNA. ( B ) The amount of IL-8 in the culture medium determined by ELISA. Data are expressed as the mean ± S.E. of three different experiments. ( C ) Western blot analysis for whole-cell extracts developed with anti-OH antibody. Actin was used as a loading control. ( D ) Cellular ROS levels determined by measuring the level of fluorescent DCF. The level of intracellular ROS is expressed as the relative increase. The value for cells without H. pylori infection in the absence of α-LA is set as 100%. * p
Figure Legend Snippet: Determination of the impact of the Nrf2 inhibitor trigonelline on IL-8 gene expression and ROS levels in H. pylori -infected AGS cells treated with α-LA. The cells were treated with 5 μM α-LA and 1 μM trigonelline for 8 h, and then infected with H. pylori . ( A ) The amount of IL-8 mRNA determined by real-time PCR analysis and normalized to cellular actin mRNA. ( B ) The amount of IL-8 in the culture medium determined by ELISA. Data are expressed as the mean ± S.E. of three different experiments. ( C ) Western blot analysis for whole-cell extracts developed with anti-OH antibody. Actin was used as a loading control. ( D ) Cellular ROS levels determined by measuring the level of fluorescent DCF. The level of intracellular ROS is expressed as the relative increase. The value for cells without H. pylori infection in the absence of α-LA is set as 100%. * p

Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

40) Product Images from "Methylation of the KEAP1 gene promoter region in human colorectal cancer"

Article Title: Methylation of the KEAP1 gene promoter region in human colorectal cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-12-66

Expression of the Nrf2 target genes NQO- 1 and AKR1C1 after t-BHQ treatment . Real-time PCR analysis of the Nrf2 target genes NQO-1 ( A , left) and AKRC1 ( B , left) in HT29 cells (methylated) and Colo320DM cells (unmethylated). Cells were treated with the Keap1 stimulator t-BHQ for 24 h. Columns, mean ( n = 3); bars, SD. * P
Figure Legend Snippet: Expression of the Nrf2 target genes NQO- 1 and AKR1C1 after t-BHQ treatment . Real-time PCR analysis of the Nrf2 target genes NQO-1 ( A , left) and AKRC1 ( B , left) in HT29 cells (methylated) and Colo320DM cells (unmethylated). Cells were treated with the Keap1 stimulator t-BHQ for 24 h. Columns, mean ( n = 3); bars, SD. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Methylation

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Article Snippet: .. Moreover, the number of cells in Nrf2 overexpressing middle-aged grafts was also higher (although not significantly, p > 0.05) compared with control middle-aged grafts transduced with just eGFP ( ). ..

Transfection:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: .. Under these conditions, interestingly the survival of the cells ( ) was not significantly affected however, the proliferation substantially improved ( , p < 0.001, untreated versus Nrf2 transfected). .. We additionally also assessed DG NSPCs from newborn (postnatal day 0) Nrf2 knockout (Nrf2-/-) and WT (Nrf2+/+) mice.

other:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
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Expressing:

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Mouse Assay:

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    Santa Cruz Biotechnology antibodies against keap1
    The interaction between p62 and <t>Keap1</t> and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control
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    The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Stable Transfection, Multiple Displacement Amplification, Plasmid Preparation

    p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Expressing, Plasmid Preparation

    p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Transfection, Expressing, Plasmid Preparation

    Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques:

    Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    doi: 10.1155/2015/187265

    Figure Lengend Snippet: Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    doi: 10.1155/2015/187265

    Figure Lengend Snippet: Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    doi: 10.1155/2015/187265

    Figure Lengend Snippet: Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques:

    Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Nuclear Magnetic Resonance, Labeling, Cell Culture, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Western Blot, Isolation, Incubation

    Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Western Blot, Affinity Purification, Concentration Assay, Sample Prep, Expressing, Quantitative RT-PCR, shRNA, Incubation, SDS Page, Silver Staining, Purification

    Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Molecular Weight, Western Blot, Expressing, Migration, Incubation, In Vitro, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitation Assay, Activity Assay, shRNA

    Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Modification, Mutagenesis, Nuclear Magnetic Resonance, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Activation Assay

    MS2 analysis of CR-MGx crosslinked KEAP1 peptide. a, Targeted Parallel reaction monitoring (PRM) transitions ( n =6). b, Annotated MS2 spectrum from the crosslinked C151-R135 KEAP1 peptide.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: MS2 analysis of CR-MGx crosslinked KEAP1 peptide. a, Targeted Parallel reaction monitoring (PRM) transitions ( n =6). b, Annotated MS2 spectrum from the crosslinked C151-R135 KEAP1 peptide.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques:

    Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.

    Journal: Oncotarget

    Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

    doi:

    Figure Lengend Snippet: Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.

    Article Snippet: Protein was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA) and probed with anti-LC3-II (Novus Biologicals, Littleton, CO), anti-Beclin-1 (Novus Biologicals, Littleton, CO), anti-Atg7 (Sigma, St. Louis, MO), anti-p62 (BioLegend, San Diego, CA) or anti-Keap1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.

    Techniques: Activity Assay, Multiple Displacement Amplification, Marker, Positive Control, Binding Assay

    Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p

    Journal: Oncotarget

    Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

    doi:

    Figure Lengend Snippet: Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p

    Article Snippet: Protein was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA) and probed with anti-LC3-II (Novus Biologicals, Littleton, CO), anti-Beclin-1 (Novus Biologicals, Littleton, CO), anti-Atg7 (Sigma, St. Louis, MO), anti-p62 (BioLegend, San Diego, CA) or anti-Keap1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.

    Techniques: Multiple Displacement Amplification, Transfection, Western Blot, Positive Control