Structured Review

Santa Cruz Biotechnology anti ho 1
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 "Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome"

Article Title: Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2019.00254

eIF4GI and eIF4GII, but not DAP5, are degraded under oxidative stress. (A) NIH-3T3 cells were untreated or treated with increasing concentration of H 2 O 2 in the presence or absence of lactacystin (described in Alard et al., 2009 ), and protein extracts were subjected to western-blotting as indicated. (B) NIH-3T3 cell extracts were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with either eIF4GI or eIF4GII antibodies (left). NIH-3T3 extracts of cells either untransfected of transfected with NQO1 cDNA were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with NQO1 antibodies (right). (C) NIH-3T3 cells were untreated or treated with 300 μM dicumarol (Dic) at different times and proteins were visualized by western-blotting as indicated. (D) Following transfection with HA-tagged, full-length or N-terminal cDNAs, NIH-3T3 cells were untreated or treated with 300 μM dicumarol for 8 h and proteins visualized by western-blotting as indicated.
Figure Legend Snippet: eIF4GI and eIF4GII, but not DAP5, are degraded under oxidative stress. (A) NIH-3T3 cells were untreated or treated with increasing concentration of H 2 O 2 in the presence or absence of lactacystin (described in Alard et al., 2009 ), and protein extracts were subjected to western-blotting as indicated. (B) NIH-3T3 cell extracts were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with either eIF4GI or eIF4GII antibodies (left). NIH-3T3 extracts of cells either untransfected of transfected with NQO1 cDNA were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with NQO1 antibodies (right). (C) NIH-3T3 cells were untreated or treated with 300 μM dicumarol (Dic) at different times and proteins were visualized by western-blotting as indicated. (D) Following transfection with HA-tagged, full-length or N-terminal cDNAs, NIH-3T3 cells were untreated or treated with 300 μM dicumarol for 8 h and proteins visualized by western-blotting as indicated.

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

Induction of NRF2 and NQO1 proteins under oxidative stress is independent of DAP5. Protein extracts of stably transfected NIH-3T3 cells grown in the absence or presence of doxycycline (Dox) for 48 h and untreated or treated with 1 mM H 2 O 2 for 4 h were subjected to western-blotting with the indicated antibodies. The bottom-to-top α–β–γ symbols denote hypo- to hyperphosphorylated 4E-BP1 isoforms.
Figure Legend Snippet: Induction of NRF2 and NQO1 proteins under oxidative stress is independent of DAP5. Protein extracts of stably transfected NIH-3T3 cells grown in the absence or presence of doxycycline (Dox) for 48 h and untreated or treated with 1 mM H 2 O 2 for 4 h were subjected to western-blotting with the indicated antibodies. The bottom-to-top α–β–γ symbols denote hypo- to hyperphosphorylated 4E-BP1 isoforms.

Techniques Used: Stable Transfection, Transfection, Western Blot

3) Product Images from "Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses"

Article Title: Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses

Journal: Redox Biology

doi: 10.1016/j.redox.2015.12.006

The protective effects of CA, QU and AV on UVA-mediated downregulation of Nrf2 target genes. (A) γ-GCL (γ-GCLC and γ-GCLM), (B) GST and (C) NQO1 mRNA expressions were assessed by real-time RT-PCR analysis at 2 h after UVA irradiation in B16F10 cells pretreated with test compounds. The statistical significance of differences between the control and irradiated cells was evaluated by Student’s t test and between UVA-irradiated and compounds-treated cells by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ## P
Figure Legend Snippet: The protective effects of CA, QU and AV on UVA-mediated downregulation of Nrf2 target genes. (A) γ-GCL (γ-GCLC and γ-GCLM), (B) GST and (C) NQO1 mRNA expressions were assessed by real-time RT-PCR analysis at 2 h after UVA irradiation in B16F10 cells pretreated with test compounds. The statistical significance of differences between the control and irradiated cells was evaluated by Student’s t test and between UVA-irradiated and compounds-treated cells by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ## P

Techniques Used: Quantitative RT-PCR, Irradiation

4) Product Images from "Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells"

Article Title: Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells

Journal: British Journal of Cancer

doi: 10.1038/bjc.2014.380

ATO induced Nrf2 targets in AML cells. ( A ) HL60 and THP-1 nuclear (N) or total (T) protein levels of Nrf2, TBP1, HO-1, NQO1 and β -actin. Cells were incubated for 24 h with 6.25 μ M ATO. ( B ) Reverse-transcription PCR (upper panels) analysis of GAPDH, HO-1, NQO1, GCLM and ferritin targets mRNA levels and nuclear Nrf2 translocation (bottom panels) in HL60 and THP-1 cells following ATO (6.25 μ M ) or t -BHQ (25 μ M ) treatments for 24 h. ( C ) HL60 cells were electroporated either with the pRS-control or pRS-shNrf2 plasmids and treated with ATO (6.25 μ M ) for 12 h. Total protein extracts were analysed for the presence of Nrf2, NQO1, HO-1 and β -actin by immunoblotting. ( D ) HL60 and THP-1 cells were treated with different concentrations of ATO, Ara-C or daunorubicin for 24 h, as indicated. Following treatment, total protein extracts were prepared and NQO1, HO-1 and β -actin protein levels were evaluated by immunoblotting. A typical western blot out of three experiments is shown. ( E ) GSH content was determined in HL60 and THP-1 cells treated with ATO (6.25 μ M ), Ara-C (5 μ M ), daunorubicin (0.5 μ M ) or t -BHQ (25 μ M ) for 24 h. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n= 3; * P ⩽0.05, ** P ⩽0.01, *** P ⩽0.001). Ctl, control.
Figure Legend Snippet: ATO induced Nrf2 targets in AML cells. ( A ) HL60 and THP-1 nuclear (N) or total (T) protein levels of Nrf2, TBP1, HO-1, NQO1 and β -actin. Cells were incubated for 24 h with 6.25 μ M ATO. ( B ) Reverse-transcription PCR (upper panels) analysis of GAPDH, HO-1, NQO1, GCLM and ferritin targets mRNA levels and nuclear Nrf2 translocation (bottom panels) in HL60 and THP-1 cells following ATO (6.25 μ M ) or t -BHQ (25 μ M ) treatments for 24 h. ( C ) HL60 cells were electroporated either with the pRS-control or pRS-shNrf2 plasmids and treated with ATO (6.25 μ M ) for 12 h. Total protein extracts were analysed for the presence of Nrf2, NQO1, HO-1 and β -actin by immunoblotting. ( D ) HL60 and THP-1 cells were treated with different concentrations of ATO, Ara-C or daunorubicin for 24 h, as indicated. Following treatment, total protein extracts were prepared and NQO1, HO-1 and β -actin protein levels were evaluated by immunoblotting. A typical western blot out of three experiments is shown. ( E ) GSH content was determined in HL60 and THP-1 cells treated with ATO (6.25 μ M ), Ara-C (5 μ M ), daunorubicin (0.5 μ M ) or t -BHQ (25 μ M ) for 24 h. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n= 3; * P ⩽0.05, ** P ⩽0.01, *** P ⩽0.001). Ctl, control.

Techniques Used: Incubation, Polymerase Chain Reaction, Translocation Assay, Acetylene Reduction Assay, Western Blot, CTL Assay

5) Product Images from "NAD(P)H: Quinone Oxidoreductase 1 Deficiency Conjoint with Marginal Vitamin C Deficiency Causes Cigarette Smoke Induced Myelodysplastic Syndromes"

Article Title: NAD(P)H: Quinone Oxidoreductase 1 Deficiency Conjoint with Marginal Vitamin C Deficiency Causes Cigarette Smoke Induced Myelodysplastic Syndromes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020590

NQO1 Activity of bone marrow cells of guinea pigs fed 0.5 mg or 15 mg vit C/day. (Panel A) AIR, exposed to air; DC, fed 3 mg DC/day; CS, exposed to CS; DC+CS, fed 3 mg DC/day and exposed to CS. * indicates significant difference (p
Figure Legend Snippet: NQO1 Activity of bone marrow cells of guinea pigs fed 0.5 mg or 15 mg vit C/day. (Panel A) AIR, exposed to air; DC, fed 3 mg DC/day; CS, exposed to CS; DC+CS, fed 3 mg DC/day and exposed to CS. * indicates significant difference (p

Techniques Used: Activity Assay

MDS produced in the guinea pigs are irreversible. (Panel A ) Differential staining showing persistent changes in blood and bone marrow cell morphology of MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. A, a–d , represent sham controls (fed 0.5 mg vitamin C/day and exposed to air); A, e–h , represent MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. Blood smear - Leishman stain; bone marrow aspirate - Wright Geimsa stain, except Perls' stain in d and h ; (magnification 400×). (Panel B ) Measurement of CD34(+) cells in bone marrow by flow cytometry. (Panel C ) Geimsa-stained metaphase spread showing aneuploidy in MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day; (magnification 1000×). (Panel D ) NQO1 Activity of bone marrow cells. Bars over the respective columns represent means ± SD (n = 4); * indicates significant difference (p
Figure Legend Snippet: MDS produced in the guinea pigs are irreversible. (Panel A ) Differential staining showing persistent changes in blood and bone marrow cell morphology of MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. A, a–d , represent sham controls (fed 0.5 mg vitamin C/day and exposed to air); A, e–h , represent MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. Blood smear - Leishman stain; bone marrow aspirate - Wright Geimsa stain, except Perls' stain in d and h ; (magnification 400×). (Panel B ) Measurement of CD34(+) cells in bone marrow by flow cytometry. (Panel C ) Geimsa-stained metaphase spread showing aneuploidy in MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day; (magnification 1000×). (Panel D ) NQO1 Activity of bone marrow cells. Bars over the respective columns represent means ± SD (n = 4); * indicates significant difference (p

Techniques Used: Produced, Staining, Leishman Stain, Flow Cytometry, Cytometry, Activity Assay

6) 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

7) Product Images from "The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1"

Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01672-12

NQO1 inhibits PGC-1α proteasomal degradation and increases its protein half-life. (A) HEK-293T cells and HEK-293T cells stably expressing the Flag-tagged β4 (PSMB2) ring subunit were transiently transfected with untagged PGC-1α-
Figure Legend Snippet: NQO1 inhibits PGC-1α proteasomal degradation and increases its protein half-life. (A) HEK-293T cells and HEK-293T cells stably expressing the Flag-tagged β4 (PSMB2) ring subunit were transiently transfected with untagged PGC-1α-

Techniques Used: Pyrolysis Gas Chromatography, Stable Transfection, Expressing, Transfection

NQO1 knockdown in myoblasts reduces steady-state PGC-1α protein levels and activity. (A) NQO1 was knocked down in C2C12 cells using a lentivirus-based approach (3 individual experiments); all were run on the same gel, and cells were analyzed for
Figure Legend Snippet: NQO1 knockdown in myoblasts reduces steady-state PGC-1α protein levels and activity. (A) NQO1 was knocked down in C2C12 cells using a lentivirus-based approach (3 individual experiments); all were run on the same gel, and cells were analyzed for

Techniques Used: Pyrolysis Gas Chromatography, Activity Assay

NQO1 protects PGC-1α by NADH-dependent interaction. (A) HEK-293 cells were transfected as indicated and harvested for analysis 24 h posttransfection. HA beads were used to immunoprecipitate (IP) HA-tagged PGC-1α, whereas HA-p73β
Figure Legend Snippet: NQO1 protects PGC-1α by NADH-dependent interaction. (A) HEK-293 cells were transfected as indicated and harvested for analysis 24 h posttransfection. HA beads were used to immunoprecipitate (IP) HA-tagged PGC-1α, whereas HA-p73β

Techniques Used: Pyrolysis Gas Chromatography, Transfection

PGC-1α is an intrinsically disordered protein by prediction, susceptible to in vitro degradation by the 20S PC and protected by NQO1. (A) Analysis of PGC-1α and PCNA amino acid sequences by the FoldIndex prediction program. (B) In vitro
Figure Legend Snippet: PGC-1α is an intrinsically disordered protein by prediction, susceptible to in vitro degradation by the 20S PC and protected by NQO1. (A) Analysis of PGC-1α and PCNA amino acid sequences by the FoldIndex prediction program. (B) In vitro

Techniques Used: Pyrolysis Gas Chromatography, In Vitro

Schematic model illustrating the convergent actions of CREB, NQO1, AMPK, and Sirt1 on PGC-1α. The scheme summarizes PGC-1α transcription and posttranslational regulation by different known metabolite-sensing proteins, including NQO1.
Figure Legend Snippet: Schematic model illustrating the convergent actions of CREB, NQO1, AMPK, and Sirt1 on PGC-1α. The scheme summarizes PGC-1α transcription and posttranslational regulation by different known metabolite-sensing proteins, including NQO1.

Techniques Used: Pyrolysis Gas Chromatography

A role for NQO1 activity in regulating PGC-1α in fasting liver. (A) Mice were either fed ad libitum or fasted during the indicated time points (starting from the beginning of the night phase) and then sacrificed for hepatic analysis of NADH and
Figure Legend Snippet: A role for NQO1 activity in regulating PGC-1α in fasting liver. (A) Mice were either fed ad libitum or fasted during the indicated time points (starting from the beginning of the night phase) and then sacrificed for hepatic analysis of NADH and

Techniques Used: Activity Assay, Pyrolysis Gas Chromatography, Mouse Assay

NQO1 and PGC-1α steady-state levels in myoblasts and myotubes. (A) Analysis of PGC-1α and NQO1 protein and mRNA levels in C2C12 myoblast (MB) cells and at sequential days after the cells were put in differentiation medium. (B) Same as
Figure Legend Snippet: NQO1 and PGC-1α steady-state levels in myoblasts and myotubes. (A) Analysis of PGC-1α and NQO1 protein and mRNA levels in C2C12 myoblast (MB) cells and at sequential days after the cells were put in differentiation medium. (B) Same as

Techniques Used: Pyrolysis Gas Chromatography

NQO1 regulates PGC-1α accumulation in response to induction by starvation-mimicking conditions in mouse primary hepatocytes. (A) Primary hepatocytes were infected with either GFP- or NQO1-expressing adenoviruses. Expression levels of NQO1 mRNA
Figure Legend Snippet: NQO1 regulates PGC-1α accumulation in response to induction by starvation-mimicking conditions in mouse primary hepatocytes. (A) Primary hepatocytes were infected with either GFP- or NQO1-expressing adenoviruses. Expression levels of NQO1 mRNA

Techniques Used: Pyrolysis Gas Chromatography, Infection, Expressing

8) Product Images from "Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis"

Article Title: Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2014.41

Nrf2 playing a key role in the mechanism of anti-hepatic fibrosis by Rg1. (A) Effect of Rg1 on prolieration in HSCs. Primary HSCs were incubated with Rg1 and CCl 4 (10 mmol/L) for 24 h. Data represent mean±SD of 6 separate experiments. (B) Nrf2 siRNA inhibits Rg1-induced translocation of Nrf2. Cells were treated with 1 μmol/L Rg1 for 24 h after control or Nrf2 siRNA transfection for 48 h. Protein expression of Nrf2 was detected by Western blotting. Representative blots were from three independent experiments. The content of MDA (C), GPX (D), PCIII (E), and HA (F) in the cell cultured supernatant and the α-SMA expression in HSCs (G). Cells were treated with 1 μmol/L Rg1 and CCl 4 (10 mmol/L) or DMSO for 24 h after control or Nrf2 siRNA transfection for 48 h; non-siRNA groups were cultured for 48 h and then treated with 1 μmol/L Rg1 and CCl 4 (10 mmol/L) or DMSO for 24 h. The data were expressed as mean±SD. n =6. Statistical evaluation was performed using t -test. b P
Figure Legend Snippet: Nrf2 playing a key role in the mechanism of anti-hepatic fibrosis by Rg1. (A) Effect of Rg1 on prolieration in HSCs. Primary HSCs were incubated with Rg1 and CCl 4 (10 mmol/L) for 24 h. Data represent mean±SD of 6 separate experiments. (B) Nrf2 siRNA inhibits Rg1-induced translocation of Nrf2. Cells were treated with 1 μmol/L Rg1 for 24 h after control or Nrf2 siRNA transfection for 48 h. Protein expression of Nrf2 was detected by Western blotting. Representative blots were from three independent experiments. The content of MDA (C), GPX (D), PCIII (E), and HA (F) in the cell cultured supernatant and the α-SMA expression in HSCs (G). Cells were treated with 1 μmol/L Rg1 and CCl 4 (10 mmol/L) or DMSO for 24 h after control or Nrf2 siRNA transfection for 48 h; non-siRNA groups were cultured for 48 h and then treated with 1 μmol/L Rg1 and CCl 4 (10 mmol/L) or DMSO for 24 h. The data were expressed as mean±SD. n =6. Statistical evaluation was performed using t -test. b P

Techniques Used: Incubation, Translocation Assay, Transfection, Expressing, Western Blot, Multiple Displacement Amplification, Cell Culture

Effects of Rg1 on CCl 4 -induced expression of hepatic α-SMA. Representative photomicrographs of liver histology from each group are shown as follows: Control (A), CCl 4 alone (B), CCl 4 +Bicyclol (C), CCl 4 +Rg1-10 mg/kg (D), CCl 4 +Rg1-20 mg/kg (E) and CCl 4 +Rg1-40 mg/kg (F). Original magnification: ×100. Scale bar, 100 μm. (G) Morphometrical analysis was performed for evaluating percentages of α-SMA-positive area in 12 random fields per section of six animals. (H) Western bloting analysis of α-SMA in liver tissue. Representative blots were from three independent experiments. The data were expressed as means±SD. c P
Figure Legend Snippet: Effects of Rg1 on CCl 4 -induced expression of hepatic α-SMA. Representative photomicrographs of liver histology from each group are shown as follows: Control (A), CCl 4 alone (B), CCl 4 +Bicyclol (C), CCl 4 +Rg1-10 mg/kg (D), CCl 4 +Rg1-20 mg/kg (E) and CCl 4 +Rg1-40 mg/kg (F). Original magnification: ×100. Scale bar, 100 μm. (G) Morphometrical analysis was performed for evaluating percentages of α-SMA-positive area in 12 random fields per section of six animals. (H) Western bloting analysis of α-SMA in liver tissue. Representative blots were from three independent experiments. The data were expressed as means±SD. c P

Techniques Used: Expressing, Western Blot

9) Product Images from "Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma"

Article Title: Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma

Journal: Oncology Letters

doi: 10.3892/ol.2017.5821

Immunofluorescent staining of NQO1 protein in BxPc-3 cells. NQO1 protein located in the cytoplasm and nucleus of BxPc-3 cells (green indicates NQO1; blue indicates DAPI; original magnification, ×630). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1.
Figure Legend Snippet: Immunofluorescent staining of NQO1 protein in BxPc-3 cells. NQO1 protein located in the cytoplasm and nucleus of BxPc-3 cells (green indicates NQO1; blue indicates DAPI; original magnification, ×630). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1.

Techniques Used: Staining

Immunohistochemical staining of NQO1 protein expression in PDAC tissues. (A) NQO1 protein expression was evaluated in microarray of PDAC tissues. (B) NQO1 staining demonstrated negative expression in non-tumor tissues. (C) Strongly positive expression of NQO1 protein in PDAC with metastasis. (D) NQO1 protein was weakly positive expressed in PDAC. (E) NQO1 protein was negatively expressed in PDAC (Original magnification, ×200 in B-E). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1; PDAC, pancreatic ductal adenocarcinoma.
Figure Legend Snippet: Immunohistochemical staining of NQO1 protein expression in PDAC tissues. (A) NQO1 protein expression was evaluated in microarray of PDAC tissues. (B) NQO1 staining demonstrated negative expression in non-tumor tissues. (C) Strongly positive expression of NQO1 protein in PDAC with metastasis. (D) NQO1 protein was weakly positive expressed in PDAC. (E) NQO1 protein was negatively expressed in PDAC (Original magnification, ×200 in B-E). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1; PDAC, pancreatic ductal adenocarcinoma.

Techniques Used: Immunohistochemistry, Staining, Expressing, Microarray

Kaplan-Meier survival curves of PDAC patients in early and late stage. (A) Kaplan-Meier curves for OS in early-stage PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P
Figure Legend Snippet: Kaplan-Meier survival curves of PDAC patients in early and late stage. (A) Kaplan-Meier curves for OS in early-stage PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P

Techniques Used: Expressing

Kaplan-Meier survival curves illustrating the significance of NQO1 expression in PDAC patients with different grading. (A) OS rates of patients with high (solid; n=83) and low (dashed; n=43) NQO1 expression (P
Figure Legend Snippet: Kaplan-Meier survival curves illustrating the significance of NQO1 expression in PDAC patients with different grading. (A) OS rates of patients with high (solid; n=83) and low (dashed; n=43) NQO1 expression (P

Techniques Used: Expressing

Association between NQO1 expression and clinicopathological significance of PDAC. The expression level of NQO1 protein was significantly associated with (A) grading (P
Figure Legend Snippet: Association between NQO1 expression and clinicopathological significance of PDAC. The expression level of NQO1 protein was significantly associated with (A) grading (P

Techniques Used: Expressing

Kaplan-Meier survival curves of PDAC patients with lymph node metastasis and without metastasis. (A) Kaplan-Meier curves for OS in the absence of LN metastasis in PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P
Figure Legend Snippet: Kaplan-Meier survival curves of PDAC patients with lymph node metastasis and without metastasis. (A) Kaplan-Meier curves for OS in the absence of LN metastasis in PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P

Techniques Used: Expressing

10) Product Images from "Molecular responses to therapeutic proteasome inhibitors in multiple myeloma patients are donor-, cell type- and drug-dependent"

Article Title: Molecular responses to therapeutic proteasome inhibitors in multiple myeloma patients are donor-, cell type- and drug-dependent

Journal: Oncotarget

doi: 10.18632/oncotarget.24882

Differential genomic responses in PBMCs twenty four hrs post-treatment (T1) of MM patients with BTZ or CFZ Relative expression levels of the PSMB1 , PSMB2 , PSMB5 , RPN6 , RPN11 (proteasome), BECN1 , SQSTM1 , HDAC6 (autophagic/aggresomes removal), NQO1 , TXNRD1 (antioxidant), HSP27 , HSP90 , CLU (chaperone), DDIT3 (UPR ER ) and PCK2 (metabolic) genes in PBMCs isolated from MM patients 24 hrs post-treatment with therapeutic doses of BTZ or CFZ (T1; see Supplementary Figure 2 ). Inserts show immunoblotting analyses of PBMCs samples isolated from indicated MM patients at T1; protein samples were probed with antibodies against proteasomal β5 or NQO1. Normalization of gene expression was vs . basal gene expression values (set to 1) found in samples isolated before treatment initiation (T1B). ACTIN or GAPDH probing and GAPDH gene expression were used as reference for total protein and total RNA input, respectively. MB: Male BTZ; FB: Female BTZ; MC: Male CFZ; FC: Female CFZ. Bars, ± SD. * P
Figure Legend Snippet: Differential genomic responses in PBMCs twenty four hrs post-treatment (T1) of MM patients with BTZ or CFZ Relative expression levels of the PSMB1 , PSMB2 , PSMB5 , RPN6 , RPN11 (proteasome), BECN1 , SQSTM1 , HDAC6 (autophagic/aggresomes removal), NQO1 , TXNRD1 (antioxidant), HSP27 , HSP90 , CLU (chaperone), DDIT3 (UPR ER ) and PCK2 (metabolic) genes in PBMCs isolated from MM patients 24 hrs post-treatment with therapeutic doses of BTZ or CFZ (T1; see Supplementary Figure 2 ). Inserts show immunoblotting analyses of PBMCs samples isolated from indicated MM patients at T1; protein samples were probed with antibodies against proteasomal β5 or NQO1. Normalization of gene expression was vs . basal gene expression values (set to 1) found in samples isolated before treatment initiation (T1B). ACTIN or GAPDH probing and GAPDH gene expression were used as reference for total protein and total RNA input, respectively. MB: Male BTZ; FB: Female BTZ; MC: Male CFZ; FC: Female CFZ. Bars, ± SD. * P

Techniques Used: Expressing, Isolation

11) Product Images from "Baicalin Ameliorates Liver Injury Induced by Chronic plus Binge Ethanol Feeding by Modulating Oxidative Stress and Inflammation via CYP2E1 and NRF2 in Mice"

Article Title: Baicalin Ameliorates Liver Injury Induced by Chronic plus Binge Ethanol Feeding by Modulating Oxidative Stress and Inflammation via CYP2E1 and NRF2 in Mice

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2017/4820414

Baicalin prevents chronic-binge ethanol-induced liver injury by modulating NRF2 and its target genes HO-1 and NQO1. (a) Western blot analyses of nuclear extracts of NRF2 along with loading control histone H3. Hepatic mRNA of HO-1 and NQO1 was analyzed by real-time PCR. The results are expressed as fold change relative to the pair-fed group. Values represent means ± SEM and n = 6/group. ∗ P
Figure Legend Snippet: Baicalin prevents chronic-binge ethanol-induced liver injury by modulating NRF2 and its target genes HO-1 and NQO1. (a) Western blot analyses of nuclear extracts of NRF2 along with loading control histone H3. Hepatic mRNA of HO-1 and NQO1 was analyzed by real-time PCR. The results are expressed as fold change relative to the pair-fed group. Values represent means ± SEM and n = 6/group. ∗ P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction

12) Product Images from "CB1 receptor blockade ameliorates hepatic fat infiltration and inflammation and increases Nrf2-AMPK pathway in a rat model of severely uncontrolled diabetes"

Article Title: CB1 receptor blockade ameliorates hepatic fat infiltration and inflammation and increases Nrf2-AMPK pathway in a rat model of severely uncontrolled diabetes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0206152

Effects of rimonabant on Nrf2 and its downstream gene expression and AMPK phosphorylation in rat livers. (A) Hepatic Nrf2 and antioxidant-responsive element (ARE)-mediated NQO1, HO-1, GSTA, and TRNRD1 gene expression were determined by RT-PCR, normalized for all samples to ribosomal RNA (18S) level, and expressed as fold change compared to LETO control rats (LETO Con). Representative western blots for NQO1, HO-1, and β-actin (B) and p-AMPK, AMPK, and β-actin (D). The density of signal was quantified and normalized by β-actin (C) or AMPK (E). Data are expressed as mean ± SEM (n = 4–5 per group). *P
Figure Legend Snippet: Effects of rimonabant on Nrf2 and its downstream gene expression and AMPK phosphorylation in rat livers. (A) Hepatic Nrf2 and antioxidant-responsive element (ARE)-mediated NQO1, HO-1, GSTA, and TRNRD1 gene expression were determined by RT-PCR, normalized for all samples to ribosomal RNA (18S) level, and expressed as fold change compared to LETO control rats (LETO Con). Representative western blots for NQO1, HO-1, and β-actin (B) and p-AMPK, AMPK, and β-actin (D). The density of signal was quantified and normalized by β-actin (C) or AMPK (E). Data are expressed as mean ± SEM (n = 4–5 per group). *P

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

13) Product Images from "Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin"

Article Title: Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin

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

doi: 10.1073/pnas.0501828102

Curcumin inhibits p53 accumulation and p53-dependent apoptosis in γ-irradiated normal thymocytes. ( A ) Nonirradiated or 4-Gy γ-irradiated (IR) thymocytes were cultured for 5 h without (–) or with the indicated concentrations of
Figure Legend Snippet: Curcumin inhibits p53 accumulation and p53-dependent apoptosis in γ-irradiated normal thymocytes. ( A ) Nonirradiated or 4-Gy γ-irradiated (IR) thymocytes were cultured for 5 h without (–) or with the indicated concentrations of

Techniques Used: Irradiation, Cell Culture

Curcumin induces ubiquitin-independent degradation of p53 and inhibits p53-induced apoptosis in leukemic cells. ( A ) M1-t-p53 myeloid leukemic cells were cultured for 5 h at 32°C without (–) or with the indicated concentrations of curcumin
Figure Legend Snippet: Curcumin induces ubiquitin-independent degradation of p53 and inhibits p53-induced apoptosis in leukemic cells. ( A ) M1-t-p53 myeloid leukemic cells were cultured for 5 h at 32°C without (–) or with the indicated concentrations of curcumin

Techniques Used: Cell Culture

Regulation of degradation of WT p53 and the hot-spot p53 R273H mutant. ( A ) p53-null HCT116 cells were transfected with pRc/CMV human WT p53 or p53 R273H mutant. After transfection (24 h), cells were cultured for 5 h without (–) or with 300 μM
Figure Legend Snippet: Regulation of degradation of WT p53 and the hot-spot p53 R273H mutant. ( A ) p53-null HCT116 cells were transfected with pRc/CMV human WT p53 or p53 R273H mutant. After transfection (24 h), cells were cultured for 5 h without (–) or with 300 μM

Techniques Used: Mutagenesis, Transfection, Cell Culture

14) Product Images from "In vivo delivery of transcription factors with multifunctional oligonucleotides"

Article Title: In vivo delivery of transcription factors with multifunctional oligonucleotides

Journal: Nature materials

doi: 10.1038/nmat4269

DARTs are able to deliver Nrf2 to hepatocytes, up-regulate Nrf2 downstream genes, and can protect hepatocytes against reactive oxygen species (ROS) a , Nrf2 delivered by DARTs enhances the expression of HO1, NQO1, and GCLC. RT-PCR of HepG2 cells treated with DART-Nrf2 complexes up-regulate HO1, NQO1, and GCLC. Free Nrf2 had no effect on HO1, NQO1, and GCLC gene expression. b , Nrf2 delivered by DARTs reduces ROS levels in HepG2 cells stressed with hydrogen peroxide. ROS levels in hydrogen peroxide stressed cells were not significantly reduced by free Nrf2, whereas DART-Nrf2 decreased ROS production down to levels comparable to control cells, mean ± S.E, n=9. **, p
Figure Legend Snippet: DARTs are able to deliver Nrf2 to hepatocytes, up-regulate Nrf2 downstream genes, and can protect hepatocytes against reactive oxygen species (ROS) a , Nrf2 delivered by DARTs enhances the expression of HO1, NQO1, and GCLC. RT-PCR of HepG2 cells treated with DART-Nrf2 complexes up-regulate HO1, NQO1, and GCLC. Free Nrf2 had no effect on HO1, NQO1, and GCLC gene expression. b , Nrf2 delivered by DARTs reduces ROS levels in HepG2 cells stressed with hydrogen peroxide. ROS levels in hydrogen peroxide stressed cells were not significantly reduced by free Nrf2, whereas DART-Nrf2 decreased ROS production down to levels comparable to control cells, mean ± S.E, n=9. **, p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

15) Product Images from "Sulforaphane prevents microcystin-LR-induced oxidative damage and apoptosis in BALB/c mice"

Article Title: Sulforaphane prevents microcystin-LR-induced oxidative damage and apoptosis in BALB/c mice

Journal: Toxicology and applied pharmacology

doi: 10.1016/j.taap.2011.05.011

SFN stabilizes Nrf2 in vivo and inhibits lipid peroxidation and GSH reduction. (A) Immunoblot illustrating the increased expression of endogenous Nrf2 in liver tissue following i.p. injection of SFN. Mice were i.p. injected with either DMSO (lanes 1 and
Figure Legend Snippet: SFN stabilizes Nrf2 in vivo and inhibits lipid peroxidation and GSH reduction. (A) Immunoblot illustrating the increased expression of endogenous Nrf2 in liver tissue following i.p. injection of SFN. Mice were i.p. injected with either DMSO (lanes 1 and

Techniques Used: In Vivo, Expressing, Injection, Mouse Assay

16) Product Images from "Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin"

Article Title: Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin

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

doi: 10.1073/pnas.0501828102

Curcumin inhibits NQO1 enzymatic activity in vitro and in cells. Enzymatic activity of recombinant NQO1 was determined in the absence (–) or presence (+) of BSA without (–) or with the indicated concentrations of curcumin ( A ) or dicoumarol
Figure Legend Snippet: Curcumin inhibits NQO1 enzymatic activity in vitro and in cells. Enzymatic activity of recombinant NQO1 was determined in the absence (–) or presence (+) of BSA without (–) or with the indicated concentrations of curcumin ( A ) or dicoumarol

Techniques Used: Activity Assay, In Vitro, Recombinant

17) Product Images from "Corynoline Isolated from Corydalis bungeana Turcz. Exhibits Anti-Inflammatory Effects via Modulation of Nfr2 and MAPKs"

Article Title: Corynoline Isolated from Corydalis bungeana Turcz. Exhibits Anti-Inflammatory Effects via Modulation of Nfr2 and MAPKs

Journal: Molecules

doi: 10.3390/molecules21080975

Evaluation of the different cellular signaling pathways affected by corynoline in LPS-induced RAW264.7 cells. Corynoline effectively blocked the activation of p38 MAPK and JNK signaling. RAW264.7 cells were treated with LPS (150 ng/mL) alone or in combination with corynoline (1.0 μM). The cells were harvested, and the proteins were measured after 24 h.
Figure Legend Snippet: Evaluation of the different cellular signaling pathways affected by corynoline in LPS-induced RAW264.7 cells. Corynoline effectively blocked the activation of p38 MAPK and JNK signaling. RAW264.7 cells were treated with LPS (150 ng/mL) alone or in combination with corynoline (1.0 μM). The cells were harvested, and the proteins were measured after 24 h.

Techniques Used: Activation Assay

18) Product Images from "20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway"

Article Title: 20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2015.154

Effects of 20C on the expression of apoptosis-related proteins. (A) Western blotting analysis of the levels of the Bax and Bcl-2 proteins in PC12 cells exposed to rotenone in the presence or absence of various concentrations of 20C. (B) Western blotting
Figure Legend Snippet: Effects of 20C on the expression of apoptosis-related proteins. (A) Western blotting analysis of the levels of the Bax and Bcl-2 proteins in PC12 cells exposed to rotenone in the presence or absence of various concentrations of 20C. (B) Western blotting

Techniques Used: Expressing, Western Blot

19) Product Images from "Curcumin Attenuates Oxaliplatin-Induced Liver Injury and Oxidative Stress by Activating the Nrf2 Pathway"

Article Title: Curcumin Attenuates Oxaliplatin-Induced Liver Injury and Oxidative Stress by Activating the Nrf2 Pathway

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S224318

Effects of CUR and brusatol on the expression of HO-1 and NQO1 in the liver of OXA-treated mice. ( A ) The protein expression levels of HO-1 and NQO1 in the liver tissue of each group were measured by Western blotting. HO-1( B ) and NQO1( C ) bands of each group were scanned, and the data are expressed as the fold change vs the control group. The results are presented as the mean ± standard deviation of five mice from each group. *P
Figure Legend Snippet: Effects of CUR and brusatol on the expression of HO-1 and NQO1 in the liver of OXA-treated mice. ( A ) The protein expression levels of HO-1 and NQO1 in the liver tissue of each group were measured by Western blotting. HO-1( B ) and NQO1( C ) bands of each group were scanned, and the data are expressed as the fold change vs the control group. The results are presented as the mean ± standard deviation of five mice from each group. *P

Techniques Used: Expressing, Mouse Assay, Western Blot, Standard Deviation

20) Product Images from "Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer"

Article Title: Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer

Journal: Carcinogenesis

doi: 10.1093/carcin/bgr237

Antioxidants upregulate NQO1 via NRF2 -dependent pathway. ( A ) Female ACI rats were treated with E2, Vit C, Vit C + E2, BHA and BHA + E2 for 240 days as described in the Materials and methods section. At the end of the experiment, mammary tumor and mammary
Figure Legend Snippet: Antioxidants upregulate NQO1 via NRF2 -dependent pathway. ( A ) Female ACI rats were treated with E2, Vit C, Vit C + E2, BHA and BHA + E2 for 240 days as described in the Materials and methods section. At the end of the experiment, mammary tumor and mammary

Techniques Used:

21) 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

22) 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:

23) Product Images from "Hydrogen Sulfide Alleviates Diabetic Nephropathy in a Streptozotocin-induced Diabetic Rat Model"

Article Title: Hydrogen Sulfide Alleviates Diabetic Nephropathy in a Streptozotocin-induced Diabetic Rat Model

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.596593

Representative immunoblots and densitometric analysis of Nrf2 in the nucleus and cytosol ( A ) and its downstream targets HO-1 and NQO1 ( B ). *, p
Figure Legend Snippet: Representative immunoblots and densitometric analysis of Nrf2 in the nucleus and cytosol ( A ) and its downstream targets HO-1 and NQO1 ( B ). *, p

Techniques Used: Western Blot

24) Product Images from "Leflunomide Induces NAD(P)H Quinone Dehydrogenase 1 Enzyme via the Aryl hydrocarbon Receptor in Neonatal Mice"

Article Title: Leflunomide Induces NAD(P)H Quinone Dehydrogenase 1 Enzyme via the Aryl hydrocarbon Receptor in Neonatal Mice

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2017.02.051

Leflunomide up-regulates pulmonary CYP1A1 and NQO1 expression in neonatal WT mice The lung tissues of neonatal WT mice treated i.p. with the vehicle, polyethylene glycol (PEG), or 1 (L 1), 5 (L 5), or 15 (L 15) mg/kg of leflunomide daily for 4 d were harvested to determine pulmonary CYP1A1 mRNA (A), NQO1 mRNA (B), CYP1A1 protein (C), and NQO1 (D) protein expression. Densitometric analyses wherein CYP1A1 (E) and NQO (F) band intensities were normalized to β-actin. Values are means ± SEM (n=3). One-way ANOVA showed an effect of L 15 for CYP1A1 and NQO1 mRNA expression and NQO1 protein expression, and an effect of L1, L5, or L15 for CYP1A1 protein expression. Significant differences between PEG- and leflunomide-treated animals are indicated by *, p
Figure Legend Snippet: Leflunomide up-regulates pulmonary CYP1A1 and NQO1 expression in neonatal WT mice The lung tissues of neonatal WT mice treated i.p. with the vehicle, polyethylene glycol (PEG), or 1 (L 1), 5 (L 5), or 15 (L 15) mg/kg of leflunomide daily for 4 d were harvested to determine pulmonary CYP1A1 mRNA (A), NQO1 mRNA (B), CYP1A1 protein (C), and NQO1 (D) protein expression. Densitometric analyses wherein CYP1A1 (E) and NQO (F) band intensities were normalized to β-actin. Values are means ± SEM (n=3). One-way ANOVA showed an effect of L 15 for CYP1A1 and NQO1 mRNA expression and NQO1 protein expression, and an effect of L1, L5, or L15 for CYP1A1 protein expression. Significant differences between PEG- and leflunomide-treated animals are indicated by *, p

Techniques Used: Expressing, Mouse Assay

25) Product Images from "Electrophilic properties of itaconate and derivatives regulate the IκBζ–ATF3 inflammatory axis"

Article Title: Electrophilic properties of itaconate and derivatives regulate the IκBζ–ATF3 inflammatory axis

Journal: Nature

doi: 10.1038/s41586-018-0052-z

Nrf2-independent action of DI a , Western blot of IκBζ expression in wild-type or Nrf2 −/− BMDMs treated with DI and stimulated with LPS for 1 h. b , Western blot of p62 and HO-1 in wild-type or Nrf2 −/− BMDMs treated with DI and stimulated with LPS. c , Western blot of IκBζ expression in wild-type and p62-deficient BMDMs treated with DI and stimulated with LPS. d , Western blot of IκBζ expression in wild-type and Hmox1 -deficient BMDMs treated with DI and stimulated with LPS. e , Transcriptional comparison of Nrf2 −/− and wild-type BMDMs treated with DI and GSEA statistics for unfolded protein response (UPR) and IFNα pathways. f , Pathways regulated by DI in an Nrf2-independent manner. Gene ranks, normalized enrichment score (NES), P and adjusted P (padj) are shown. g, h , Western blot of Nrf2 expression ( g ) or phosphorylated and total eIF2α ( h ) in DI-treated wild-type or Atf3 −/− BMDMs. i – k , Western blot of ATF3 in BMDMs ( i, j ) and human blood monocytes ( k ) treated with DI in combination with NAC or EtGSH and stimulated with LPS. Data are representative of three experiments ( a, g, i, j ), two experiments ( b, c, h ), one experiment ( d ) and from two donors ( k .
Figure Legend Snippet: Nrf2-independent action of DI a , Western blot of IκBζ expression in wild-type or Nrf2 −/− BMDMs treated with DI and stimulated with LPS for 1 h. b , Western blot of p62 and HO-1 in wild-type or Nrf2 −/− BMDMs treated with DI and stimulated with LPS. c , Western blot of IκBζ expression in wild-type and p62-deficient BMDMs treated with DI and stimulated with LPS. d , Western blot of IκBζ expression in wild-type and Hmox1 -deficient BMDMs treated with DI and stimulated with LPS. e , Transcriptional comparison of Nrf2 −/− and wild-type BMDMs treated with DI and GSEA statistics for unfolded protein response (UPR) and IFNα pathways. f , Pathways regulated by DI in an Nrf2-independent manner. Gene ranks, normalized enrichment score (NES), P and adjusted P (padj) are shown. g, h , Western blot of Nrf2 expression ( g ) or phosphorylated and total eIF2α ( h ) in DI-treated wild-type or Atf3 −/− BMDMs. i – k , Western blot of ATF3 in BMDMs ( i, j ) and human blood monocytes ( k ) treated with DI in combination with NAC or EtGSH and stimulated with LPS. Data are representative of three experiments ( a, g, i, j ), two experiments ( b, c, h ), one experiment ( d ) and from two donors ( k .

Techniques Used: Western Blot, Expressing

DI induces an Nrf2-independent response and inhibits the IL-6–IκBζ axis via ATF3 a , f , Western blots showing IκBζ expression in Nrf2 −/− ( a ) and Atf3 −/− ( f ) BMDMs. b , g , Cytokine levels in BMDMs treated with DI, stimulated with LPS for 4 h ( b ) and 24 h ( g ), mean ± s.e.m., n = 3 experiments. c , Genes regulated by DI independently of Nrf2. Hsp90, Ire1a and Perk are also known as Hsp84-2, Ern1 and Eif2ak3 , respectively. ‘UPS response’ indicates UPS response pathways. d , Transcriptional comparison of Atf3 −/− and wild-type BMDMs and enrichment of the Nrf2-independent DI signature. e , h , Western blots of ATF3 expression in BMDMs after DI treatment ( e ) and tolerized in the presence of BSO ( h . Statistical tests used were two-tailed t -tests. KO, knockout; NES, normalized enrichment score; UPR, unfolded protein response.
Figure Legend Snippet: DI induces an Nrf2-independent response and inhibits the IL-6–IκBζ axis via ATF3 a , f , Western blots showing IκBζ expression in Nrf2 −/− ( a ) and Atf3 −/− ( f ) BMDMs. b , g , Cytokine levels in BMDMs treated with DI, stimulated with LPS for 4 h ( b ) and 24 h ( g ), mean ± s.e.m., n = 3 experiments. c , Genes regulated by DI independently of Nrf2. Hsp90, Ire1a and Perk are also known as Hsp84-2, Ern1 and Eif2ak3 , respectively. ‘UPS response’ indicates UPS response pathways. d , Transcriptional comparison of Atf3 −/− and wild-type BMDMs and enrichment of the Nrf2-independent DI signature. e , h , Western blots of ATF3 expression in BMDMs after DI treatment ( e ) and tolerized in the presence of BSO ( h . Statistical tests used were two-tailed t -tests. KO, knockout; NES, normalized enrichment score; UPR, unfolded protein response.

Techniques Used: Western Blot, Expressing, Two Tailed Test, Knock-Out

26) 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

27) Product Images from "CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation"

Article Title: CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115600

CDDO-Me is a potent radiation countermeasure in bronchial and breast epithelial cells, and Nrf2 knockdown abrogates these radioprotective effects. (A) Normal breast and lung epithelia are radioprotected at multiple doses of CDDO-Me. Cells were treated with drug 18 hours before exposure to 3 Gy gamma IR, then seeded immediately into clonogenicity. Colonies grown for ∼14 days before fixation with 6% glutaraldehyde/0.5% crystal violet stain. Mean ± SEM of four experiments seeded in triplicate, *p
Figure Legend Snippet: CDDO-Me is a potent radiation countermeasure in bronchial and breast epithelial cells, and Nrf2 knockdown abrogates these radioprotective effects. (A) Normal breast and lung epithelia are radioprotected at multiple doses of CDDO-Me. Cells were treated with drug 18 hours before exposure to 3 Gy gamma IR, then seeded immediately into clonogenicity. Colonies grown for ∼14 days before fixation with 6% glutaraldehyde/0.5% crystal violet stain. Mean ± SEM of four experiments seeded in triplicate, *p

Techniques Used: Staining

CDDO-Me activates the Nrf2 antioxidant pathway in epithelial cells. (A) Nrf2 Pathway: Nrf2 is a transcription factor normally bound by its cytoplasmic repressor Keap1, which acts as a molecular oxidative sensor and marks Nrf2 for degradation. When there is an abundance of reactive species in the cells, Nrf2 accumulates in the cytoplasm, eventually undergoing various phosphorylation events to translocate to the nucleus and bind to Antioxidant Response Elements (AREs) in the genome, resulting in the transcription of multiple antioxidative and cyto-protective genes. CDDO-Me acts by facilitating the dissociation between Keap1 and Nrf2, leading to Nrf2 activation. (B) Chemical structure of CDDO-Me: Oleana-1,9(11)-dien-28-oicacid, 2-cyano-3,12-dioxo-, methyl ester (RTA-402; bardoxolone-methyl). (C, D) CDDO-Me increases expression of ARE-driven luciferase 18 hours after drug treatment in HBEC 3KT and HME1, respectively. Firefly ARE-luciferase normalized to renilla control (RLU). Mean ± SEM of 6 replicates, *p
Figure Legend Snippet: CDDO-Me activates the Nrf2 antioxidant pathway in epithelial cells. (A) Nrf2 Pathway: Nrf2 is a transcription factor normally bound by its cytoplasmic repressor Keap1, which acts as a molecular oxidative sensor and marks Nrf2 for degradation. When there is an abundance of reactive species in the cells, Nrf2 accumulates in the cytoplasm, eventually undergoing various phosphorylation events to translocate to the nucleus and bind to Antioxidant Response Elements (AREs) in the genome, resulting in the transcription of multiple antioxidative and cyto-protective genes. CDDO-Me acts by facilitating the dissociation between Keap1 and Nrf2, leading to Nrf2 activation. (B) Chemical structure of CDDO-Me: Oleana-1,9(11)-dien-28-oicacid, 2-cyano-3,12-dioxo-, methyl ester (RTA-402; bardoxolone-methyl). (C, D) CDDO-Me increases expression of ARE-driven luciferase 18 hours after drug treatment in HBEC 3KT and HME1, respectively. Firefly ARE-luciferase normalized to renilla control (RLU). Mean ± SEM of 6 replicates, *p

Techniques Used: Activation Assay, Expressing, Luciferase

28) 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:

29) Product Images from "An excess dietary vitamin E concentration does not influence Nrf2 signaling in the liver of rats fed either soybean oil or salmon oil"

Article Title: An excess dietary vitamin E concentration does not influence Nrf2 signaling in the liver of rats fed either soybean oil or salmon oil

Journal: Nutrition & Metabolism

doi: 10.1186/s12986-017-0225-z

Effect on protein expression of Nrf2 targets. Relative Protein expression of GPX ( a ), HO-1 ( b ) and NQO1 ( c ) in the liver of rats fed diets with either soybean oil or salmon oil with various vitamin E concentrations. Bars represent relative protein level expressed as fold of control (diet with soybean oil and 25 mg/kg vitamin E) and are means ± SD from 12 animals per group. Representative immunoblots specific to GPX ( a ), HO-1 ( b ), NQO1 ( c ) and ß-actin as respective loading control are shown for one animal per group
Figure Legend Snippet: Effect on protein expression of Nrf2 targets. Relative Protein expression of GPX ( a ), HO-1 ( b ) and NQO1 ( c ) in the liver of rats fed diets with either soybean oil or salmon oil with various vitamin E concentrations. Bars represent relative protein level expressed as fold of control (diet with soybean oil and 25 mg/kg vitamin E) and are means ± SD from 12 animals per group. Representative immunoblots specific to GPX ( a ), HO-1 ( b ), NQO1 ( c ) and ß-actin as respective loading control are shown for one animal per group

Techniques Used: Expressing, Western Blot

30) Product Images from "Citral Is Renoprotective for Focal Segmental Glomerulosclerosis by Inhibiting Oxidative Stress and Apoptosis and Activating Nrf2 Pathway in Mice"

Article Title: Citral Is Renoprotective for Focal Segmental Glomerulosclerosis by Inhibiting Oxidative Stress and Apoptosis and Activating Nrf2 Pathway in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074871

Renal nuclear Nrf2 levels, cytosolic p47 phox , NQO1 and HO-1 levels. (A) Representative Western blot showing levels of cytosolic p47 phox and NQO1 and nuclear Nrf2 in kidney tissues. (B-D) Quantification of the p47 phox /β-actin (cytosolic) ratio (B), the Nrf2/β-actin (nuclear) ratio (C), and the NQO1/β-actin (cytosolic) ratio (D). The horizontal dashed lines indicate levels in normal control mice. (E) HO-1 levels in the kidney. In B-E, the data are the mean±SEM for seven mice per group. * p
Figure Legend Snippet: Renal nuclear Nrf2 levels, cytosolic p47 phox , NQO1 and HO-1 levels. (A) Representative Western blot showing levels of cytosolic p47 phox and NQO1 and nuclear Nrf2 in kidney tissues. (B-D) Quantification of the p47 phox /β-actin (cytosolic) ratio (B), the Nrf2/β-actin (nuclear) ratio (C), and the NQO1/β-actin (cytosolic) ratio (D). The horizontal dashed lines indicate levels in normal control mice. (E) HO-1 levels in the kidney. In B-E, the data are the mean±SEM for seven mice per group. * p

Techniques Used: Western Blot, Mouse Assay

31) Product Images from "Scolopendra subspinipes mutilans attenuates neuroinflammation in symptomatic hSOD1G93A mice"

Article Title: Scolopendra subspinipes mutilans attenuates neuroinflammation in symptomatic hSOD1G93A mice

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-10-131

SSM treatment regulates the expression of CD14, HO1, and NQO1 in the brainstems and spinal cords of hSOD1 G93A mice. (A) Representative Western blot showing the expression of CD14, HO1, and NQO1 in the brainstem and spinal cord following the administration of SSM. (B) Quantification of the level of CD14/tubulin, HO1/tubulin, and NQO1/tubulin. The data are presented as the means ± SEM ( N = 4 animals/genotype). Statistical significance was assessed via t -test. *** P
Figure Legend Snippet: SSM treatment regulates the expression of CD14, HO1, and NQO1 in the brainstems and spinal cords of hSOD1 G93A mice. (A) Representative Western blot showing the expression of CD14, HO1, and NQO1 in the brainstem and spinal cord following the administration of SSM. (B) Quantification of the level of CD14/tubulin, HO1/tubulin, and NQO1/tubulin. The data are presented as the means ± SEM ( N = 4 animals/genotype). Statistical significance was assessed via t -test. *** P

Techniques Used: Expressing, Mouse Assay, Western Blot

32) Product Images from "NQO1 is Required for β-Lapachone-Mediated Downregulation of Breast-Cancer Stem-Cell Activity"

Article Title: NQO1 is Required for β-Lapachone-Mediated Downregulation of Breast-Cancer Stem-Cell Activity

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123813

Sirtuin 1 (SIRT1) is not involved in bL-NQO1-mediated cell death. ( A , using NQO1 stable cells with or without bL (2 µM) for 24 h. The cell lysate was subjected to Western blot analysis with the indicated antibodies; β-tubulin and lamin B were used for cytoplasmic and nuclear markers, respectively. ( B ) Cellular localization of SIRT1 was examined in MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) with or without bL (2 µM) for the indicated time. Images were taken under a new hybrid microscope (Echo, San Diego, CA, USA). The scale bar is 130 μm. ( C ) Cell morphologies of MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) were examined after treatment with a combination of bL (2 µM), an NQO1 inhibitor (ES 936, 1 µM), and an SIRT1 inhibitor (EX 527, 1 µM) for 24 h, as indicated. Images were taken using a new hybrid microscope (Echo, Echo, San Diego, CA, USA). The scale bar is 230 μm.
Figure Legend Snippet: Sirtuin 1 (SIRT1) is not involved in bL-NQO1-mediated cell death. ( A , using NQO1 stable cells with or without bL (2 µM) for 24 h. The cell lysate was subjected to Western blot analysis with the indicated antibodies; β-tubulin and lamin B were used for cytoplasmic and nuclear markers, respectively. ( B ) Cellular localization of SIRT1 was examined in MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) with or without bL (2 µM) for the indicated time. Images were taken under a new hybrid microscope (Echo, San Diego, CA, USA). The scale bar is 130 μm. ( C ) Cell morphologies of MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) were examined after treatment with a combination of bL (2 µM), an NQO1 inhibitor (ES 936, 1 µM), and an SIRT1 inhibitor (EX 527, 1 µM) for 24 h, as indicated. Images were taken using a new hybrid microscope (Echo, Echo, San Diego, CA, USA). The scale bar is 230 μm.

Techniques Used: Western Blot, Multiple Displacement Amplification, Clone Assay, Microscopy

33) 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:

34) 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

35) Product Images from "Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1H46R-expressing ALS mouse model"

Article Title: Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1H46R-expressing ALS mouse model

Journal: Molecular Brain

doi: 10.1186/s13041-018-0373-8

High-magnification images for the distribution of ubiquitin-positive aggregates in the spinal cord from SOD1 H46R -expressing mice. a-d Representative images of double immunostaining with Ubiquitin (green) and SQSTM1 (red) ( a ), MAP2 (green; neuron marker) and Ubiquitin (red) ( b ), Ubiquitin (green) and GFAP (red; astrocyte marker) ( c ), Ubiquitin (green) and Iba1 (red; microglia marker) ( d ) in the lumbar cord (L4–5) from SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at end-stage. The nuclei were counterstained with DAPI (blue). Scale bars = 10 μm. c Arrows indicate that ubiquitin-positive aggregates are present in GFAP-positive astrocytes. d Arrows indicate that ubiquitin-positive aggregates are surrounded by microglia
Figure Legend Snippet: High-magnification images for the distribution of ubiquitin-positive aggregates in the spinal cord from SOD1 H46R -expressing mice. a-d Representative images of double immunostaining with Ubiquitin (green) and SQSTM1 (red) ( a ), MAP2 (green; neuron marker) and Ubiquitin (red) ( b ), Ubiquitin (green) and GFAP (red; astrocyte marker) ( c ), Ubiquitin (green) and Iba1 (red; microglia marker) ( d ) in the lumbar cord (L4–5) from SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at end-stage. The nuclei were counterstained with DAPI (blue). Scale bars = 10 μm. c Arrows indicate that ubiquitin-positive aggregates are present in GFAP-positive astrocytes. d Arrows indicate that ubiquitin-positive aggregates are surrounded by microglia

Techniques Used: Expressing, Mouse Assay, Double Immunostaining, Marker

Extensive accumulation of ubiquitin-positive aggregates in the spinal cord from SOD1 H46R -expressing mice at end-stage. a-c Representative images of double immunostaining with MAP2 (green; neuron marker) and Ubiquitin (red) ( a ), Ubiquitin (green) and GFAP (red; astrocyte marker) ( b ), Ubiquitin (green) and Iba1 (red; microglia marker) ( c ) in the lumbar spinal cord (L4–5) from wild-type (WT) and SQSTM1 (SQSTM1) at 28 weeks of age, SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at end-stage. The nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. b Arrows indicate colocalization of ubiquitin-positive aggregates with GFAP-positive astrocytes. c Arrows indicate colocalization of ubiquitin-positive aggregates with Iba1-positive microglia
Figure Legend Snippet: Extensive accumulation of ubiquitin-positive aggregates in the spinal cord from SOD1 H46R -expressing mice at end-stage. a-c Representative images of double immunostaining with MAP2 (green; neuron marker) and Ubiquitin (red) ( a ), Ubiquitin (green) and GFAP (red; astrocyte marker) ( b ), Ubiquitin (green) and Iba1 (red; microglia marker) ( c ) in the lumbar spinal cord (L4–5) from wild-type (WT) and SQSTM1 (SQSTM1) at 28 weeks of age, SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at end-stage. The nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. b Arrows indicate colocalization of ubiquitin-positive aggregates with GFAP-positive astrocytes. c Arrows indicate colocalization of ubiquitin-positive aggregates with Iba1-positive microglia

Techniques Used: Expressing, Mouse Assay, Double Immunostaining, Marker

Progressive accumulation of ubiquitin/SQSTM1-positive aggregates in the anterior horn of lumbar spinal cord. a-c Representative images of double immunostaining with Ubiquitin (green) and SQSTM1 (red) in the lumbar spinal cord (L4–5) from wild-type (WT), SQSTM1 (SQSTM1), SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at 16 weeks of age (wk) ( a ), 22 wk. ( b ), and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1) ( c ). The nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. Ubiquitin-positive aggregates and SQSTM1 aggregates were observed in the anterior horn of SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at 22 wk. and end-stage. Arrows indicate ubiquitin-positive aggregates that are colocalized with SQSTM1
Figure Legend Snippet: Progressive accumulation of ubiquitin/SQSTM1-positive aggregates in the anterior horn of lumbar spinal cord. a-c Representative images of double immunostaining with Ubiquitin (green) and SQSTM1 (red) in the lumbar spinal cord (L4–5) from wild-type (WT), SQSTM1 (SQSTM1), SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at 16 weeks of age (wk) ( a ), 22 wk. ( b ), and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1) ( c ). The nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. Ubiquitin-positive aggregates and SQSTM1 aggregates were observed in the anterior horn of SOD1 H46R (H46R) and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at 22 wk. and end-stage. Arrows indicate ubiquitin-positive aggregates that are colocalized with SQSTM1

Techniques Used: Double Immunostaining, Mouse Assay

36) 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

37) Product Images from "Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene"

Article Title: Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E13-11-0666

Hemin induces phosphorylation of p66Shc at Ser-36 but no mitochondrial translocation in K562 cells. (A) Endogenous p66Shc phosphorylation at Ser-36 was detected in whole-cell lysate (Total) and mitochondrial (Mito.), cytoplasmic (Cyto.), and nuclear (Nuc.) fractions of K562 cells treated with 0 and 20 μM hemin for 24 h. IPs with anti-Shc antibody were subjected to Western blots with anti–phospho-Ser-36 p66Shc antibody (IP/WB). Equivalent protein loading of IP samples was assessed by probing the blot with anti-rabbit IgG. PBR, LDH, and lamin B1 were detected by Western blots for mitochondrial, cytoplasmic, and nuclear fraction markers. (B) Phospho–Ser-36 p66Shc protein levels were measured in K562 cells treated with 20 μM hemin for 0, 3, 6, 12, 24, and 48 h. Mitochondrial and cytoplasmic fractions were used for p66Shc IP/WB. Equal protein loading of IP samples was assessed by probing the blot with anti-rabbit IgG. TOM20 and LDH are markers of mitochondria and cytoplasm, respectively. The ratio indicates pS36-p66Shc/p66Shc in cytoplasmic fractions (0 h as 1.0).
Figure Legend Snippet: Hemin induces phosphorylation of p66Shc at Ser-36 but no mitochondrial translocation in K562 cells. (A) Endogenous p66Shc phosphorylation at Ser-36 was detected in whole-cell lysate (Total) and mitochondrial (Mito.), cytoplasmic (Cyto.), and nuclear (Nuc.) fractions of K562 cells treated with 0 and 20 μM hemin for 24 h. IPs with anti-Shc antibody were subjected to Western blots with anti–phospho-Ser-36 p66Shc antibody (IP/WB). Equivalent protein loading of IP samples was assessed by probing the blot with anti-rabbit IgG. PBR, LDH, and lamin B1 were detected by Western blots for mitochondrial, cytoplasmic, and nuclear fraction markers. (B) Phospho–Ser-36 p66Shc protein levels were measured in K562 cells treated with 20 μM hemin for 0, 3, 6, 12, 24, and 48 h. Mitochondrial and cytoplasmic fractions were used for p66Shc IP/WB. Equal protein loading of IP samples was assessed by probing the blot with anti-rabbit IgG. TOM20 and LDH are markers of mitochondria and cytoplasm, respectively. The ratio indicates pS36-p66Shc/p66Shc in cytoplasmic fractions (0 h as 1.0).

Techniques Used: Translocation Assay, Western Blot

38) Product Images from "Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway"

Article Title: Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S110505

UA sensitizes HepG2/DDP cells to low-dose cisplatin via inhibition of Nrf2/ARE signaling pathway. Notes: ( A ) HepG2/DDP cells were transfected with Nrf2 siRNA (si-Nrf2) or negative control (si-Con), or ( C ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with 8.92 μg/mL cisplatin (IC 30 of cisplatin for HepG2/DDP cells) and/or UA (2.25 μg/mL) for 48 hours. The level of Nrf2, HO-1, NQO1, and GST was detected by Western blot analysis. ( B ) HepG2/DDP cells were transfected with si-Nrf2 or si-Con, or ( D ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with series concentration of cisplatin (2–512 μg/mL) and/or UA (2.25 μg/mL) for 48 hours. The inhibition rate of cell was detected by CCK8 assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: ARE, antioxidant response element; CCK8, Cell Counting Kit 8; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin–resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; IC 30 , 30% inhibitory concentration; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; siRNA, small interfering RNA; UA, ursolic acid.
Figure Legend Snippet: UA sensitizes HepG2/DDP cells to low-dose cisplatin via inhibition of Nrf2/ARE signaling pathway. Notes: ( A ) HepG2/DDP cells were transfected with Nrf2 siRNA (si-Nrf2) or negative control (si-Con), or ( C ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with 8.92 μg/mL cisplatin (IC 30 of cisplatin for HepG2/DDP cells) and/or UA (2.25 μg/mL) for 48 hours. The level of Nrf2, HO-1, NQO1, and GST was detected by Western blot analysis. ( B ) HepG2/DDP cells were transfected with si-Nrf2 or si-Con, or ( D ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with series concentration of cisplatin (2–512 μg/mL) and/or UA (2.25 μg/mL) for 48 hours. The inhibition rate of cell was detected by CCK8 assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: ARE, antioxidant response element; CCK8, Cell Counting Kit 8; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin–resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; IC 30 , 30% inhibitory concentration; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; siRNA, small interfering RNA; UA, ursolic acid.

Techniques Used: Inhibition, Transfection, Negative Control, Plasmid Preparation, Western Blot, Concentration Assay, CCK-8 Assay, Cell Counting, Standard Deviation, Small Interfering RNA

Nrf2 was overexpressed in cisplatin-resistant human hepatocellular carcinoma HepG2/DDP cells. Notes: ( A ) HepG2 cells were treated with series concentration of cisplatin (0.1–25.6 μg/mL) for 48 hours. ( B ) HepG2/DDP cells were treated with series concentration of cisplatin (2–512 μg/mL) for 48 hours. ( C ) The level of Nrf2 and its downstream target genes HO-1, NQO1, and GST in HepG2 and HepG2/DDP cells was detected by Western blot assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2, hepatocellular carcinoma cell line; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation.
Figure Legend Snippet: Nrf2 was overexpressed in cisplatin-resistant human hepatocellular carcinoma HepG2/DDP cells. Notes: ( A ) HepG2 cells were treated with series concentration of cisplatin (0.1–25.6 μg/mL) for 48 hours. ( B ) HepG2/DDP cells were treated with series concentration of cisplatin (2–512 μg/mL) for 48 hours. ( C ) The level of Nrf2 and its downstream target genes HO-1, NQO1, and GST in HepG2 and HepG2/DDP cells was detected by Western blot assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2, hepatocellular carcinoma cell line; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation.

Techniques Used: Concentration Assay, Western Blot, Standard Deviation

UA–cisplatin combination downregulates Nrf2 and its substrates. Notes: The protein expression levels of Nrf2, HO-1, NQO1, and GST of HepG2/DDP cells treated with 8.92 μg/mL cisplatin and/or UA (2.25 μg/mL) for 48 hours were detected by Western blot analysis. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; UA, ursolic acid.
Figure Legend Snippet: UA–cisplatin combination downregulates Nrf2 and its substrates. Notes: The protein expression levels of Nrf2, HO-1, NQO1, and GST of HepG2/DDP cells treated with 8.92 μg/mL cisplatin and/or UA (2.25 μg/mL) for 48 hours were detected by Western blot analysis. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; UA, ursolic acid.

Techniques Used: Expressing, Western Blot, Standard Deviation

39) 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

40) 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

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Transduction:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
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
Article Snippet: Finally, in the pattern separation test, the Nrf2-/- animals exhibited a compromised behavior as indicated by their substantially reduced exploration of the object in the novel context (p < 0.05, ) when compared with their WT counterparts.

Article Title: Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs
Article Snippet: NRF2 (erythroid-derived factor 2) is one of the main transcription factors that determine antioxidant response of the cells to the action of the internal and external ROS.

Article Title: Nrf2-mediated anti-oxidant effects contribute to suppression of non-alcoholic steatohepatitis-associated hepatocellular carcinoma in murine model
Article Snippet: The results of this study support that Nrf2 and its related metabolites have protective effects on liver injury, inflammation, and tumorigenesis. ( , )

Expressing:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: .. Here, a significant reduction in MCM2 , Sox2 , and GFAP/nestin ( ) expressing NSPCs was noted in the DG of the Nrf2-/- mice. .. Moreover, the number of Dcx+ newborn neurons was also significantly reduced in the Nrf2-/- mice compared with WT controls ( ).

Mouse Assay:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: .. Here, a significant reduction in MCM2 , Sox2 , and GFAP/nestin ( ) expressing NSPCs was noted in the DG of the Nrf2-/- mice. .. Moreover, the number of Dcx+ newborn neurons was also significantly reduced in the Nrf2-/- mice compared with WT controls ( ).

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    Santa Cruz Biotechnology anti nqo1
    eIF4GI and eIF4GII, but not DAP5, are degraded under oxidative stress. (A) NIH-3T3 cells were untreated or treated with increasing concentration of H 2 O 2 in the presence or absence of lactacystin (described in Alard et al., 2009 ), and protein extracts were subjected to western-blotting as indicated. (B) NIH-3T3 cell extracts were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with either eIF4GI or eIF4GII antibodies (left). NIH-3T3 extracts of cells either untransfected of transfected with <t>NQO1</t> cDNA were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with NQO1 antibodies (right). (C) NIH-3T3 cells were untreated or treated with 300 μM dicumarol (Dic) at different times and proteins were visualized by western-blotting as indicated. (D) Following transfection with HA-tagged, full-length or N-terminal cDNAs, NIH-3T3 cells were untreated or treated with 300 μM dicumarol for 8 h and proteins visualized by western-blotting as indicated.
    Anti Nqo1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti nqo1/product/Santa Cruz Biotechnology
    Average 91 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    anti nqo1 - by Bioz Stars, 2020-09
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    89
    Santa Cruz Biotechnology mouse anti nqo1
    <t>NQO1</t> inhibits PGC-1α proteasomal degradation and increases its protein half-life. (A) HEK-293T cells and HEK-293T cells stably expressing the Flag-tagged β4 (PSMB2) ring subunit were transiently transfected with untagged PGC-1α-
    Mouse Anti Nqo1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti nqo1/product/Santa Cruz Biotechnology
    Average 89 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    mouse anti nqo1 - by Bioz Stars, 2020-09
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    Image Search Results


    eIF4GI and eIF4GII, but not DAP5, are degraded under oxidative stress. (A) NIH-3T3 cells were untreated or treated with increasing concentration of H 2 O 2 in the presence or absence of lactacystin (described in Alard et al., 2009 ), and protein extracts were subjected to western-blotting as indicated. (B) NIH-3T3 cell extracts were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with either eIF4GI or eIF4GII antibodies (left). NIH-3T3 extracts of cells either untransfected of transfected with NQO1 cDNA were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with NQO1 antibodies (right). (C) NIH-3T3 cells were untreated or treated with 300 μM dicumarol (Dic) at different times and proteins were visualized by western-blotting as indicated. (D) Following transfection with HA-tagged, full-length or N-terminal cDNAs, NIH-3T3 cells were untreated or treated with 300 μM dicumarol for 8 h and proteins visualized by western-blotting as indicated.

    Journal: Frontiers in Genetics

    Article Title: Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome

    doi: 10.3389/fgene.2019.00254

    Figure Lengend Snippet: eIF4GI and eIF4GII, but not DAP5, are degraded under oxidative stress. (A) NIH-3T3 cells were untreated or treated with increasing concentration of H 2 O 2 in the presence or absence of lactacystin (described in Alard et al., 2009 ), and protein extracts were subjected to western-blotting as indicated. (B) NIH-3T3 cell extracts were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with either eIF4GI or eIF4GII antibodies (left). NIH-3T3 extracts of cells either untransfected of transfected with NQO1 cDNA were subjected to western-blotting with the indicated antibodies either directly (input) or after immunoprecipitation (IP) with NQO1 antibodies (right). (C) NIH-3T3 cells were untreated or treated with 300 μM dicumarol (Dic) at different times and proteins were visualized by western-blotting as indicated. (D) Following transfection with HA-tagged, full-length or N-terminal cDNAs, NIH-3T3 cells were untreated or treated with 300 μM dicumarol for 8 h and proteins visualized by western-blotting as indicated.

    Article Snippet: The antibodies used were as follows: anti-eIF4GI and anti-eIF4GII (gifts of Prof. Nahum Sonenberg); anti-DAP5 (CliniSciences #610742); anti-HA-7 (Sigma); anti-β-tubulin (GeneTex #6288022); anti-4E-BP1, anti-NRF2 and anti-p53 (Cell Signaling Technologies #9452, #12721, and #1C12, respectively); anti-Core 20S (Enzo Life Sciences #PW8155); and anti-NQO1 (Santa Cruz #C19).

    Techniques: Concentration Assay, Western Blot, Immunoprecipitation, Transfection

    Induction of NRF2 and NQO1 proteins under oxidative stress is independent of DAP5. Protein extracts of stably transfected NIH-3T3 cells grown in the absence or presence of doxycycline (Dox) for 48 h and untreated or treated with 1 mM H 2 O 2 for 4 h were subjected to western-blotting with the indicated antibodies. The bottom-to-top α–β–γ symbols denote hypo- to hyperphosphorylated 4E-BP1 isoforms.

    Journal: Frontiers in Genetics

    Article Title: Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome

    doi: 10.3389/fgene.2019.00254

    Figure Lengend Snippet: Induction of NRF2 and NQO1 proteins under oxidative stress is independent of DAP5. Protein extracts of stably transfected NIH-3T3 cells grown in the absence or presence of doxycycline (Dox) for 48 h and untreated or treated with 1 mM H 2 O 2 for 4 h were subjected to western-blotting with the indicated antibodies. The bottom-to-top α–β–γ symbols denote hypo- to hyperphosphorylated 4E-BP1 isoforms.

    Article Snippet: The antibodies used were as follows: anti-eIF4GI and anti-eIF4GII (gifts of Prof. Nahum Sonenberg); anti-DAP5 (CliniSciences #610742); anti-HA-7 (Sigma); anti-β-tubulin (GeneTex #6288022); anti-4E-BP1, anti-NRF2 and anti-p53 (Cell Signaling Technologies #9452, #12721, and #1C12, respectively); anti-Core 20S (Enzo Life Sciences #PW8155); and anti-NQO1 (Santa Cruz #C19).

    Techniques: Stable Transfection, Transfection, Western Blot

    NQO1 Activity of bone marrow cells of guinea pigs fed 0.5 mg or 15 mg vit C/day. (Panel A) AIR, exposed to air; DC, fed 3 mg DC/day; CS, exposed to CS; DC+CS, fed 3 mg DC/day and exposed to CS. * indicates significant difference (p

    Journal: PLoS ONE

    Article Title: NAD(P)H: Quinone Oxidoreductase 1 Deficiency Conjoint with Marginal Vitamin C Deficiency Causes Cigarette Smoke Induced Myelodysplastic Syndromes

    doi: 10.1371/journal.pone.0020590

    Figure Lengend Snippet: NQO1 Activity of bone marrow cells of guinea pigs fed 0.5 mg or 15 mg vit C/day. (Panel A) AIR, exposed to air; DC, fed 3 mg DC/day; CS, exposed to CS; DC+CS, fed 3 mg DC/day and exposed to CS. * indicates significant difference (p

    Article Snippet: Supporting Information NQO1 expression in the bone marrow cells, indicating t hat there is no significant change in NQO1 expression at the protein level. (Panel A) and (Panel B) Bone marrow lysates were separated by 10% SDS PAGE and subjected to immmunobloting using antibody against NQO1.

    Techniques: Activity Assay

    MDS produced in the guinea pigs are irreversible. (Panel A ) Differential staining showing persistent changes in blood and bone marrow cell morphology of MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. A, a–d , represent sham controls (fed 0.5 mg vitamin C/day and exposed to air); A, e–h , represent MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. Blood smear - Leishman stain; bone marrow aspirate - Wright Geimsa stain, except Perls' stain in d and h ; (magnification 400×). (Panel B ) Measurement of CD34(+) cells in bone marrow by flow cytometry. (Panel C ) Geimsa-stained metaphase spread showing aneuploidy in MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day; (magnification 1000×). (Panel D ) NQO1 Activity of bone marrow cells. Bars over the respective columns represent means ± SD (n = 4); * indicates significant difference (p

    Journal: PLoS ONE

    Article Title: NAD(P)H: Quinone Oxidoreductase 1 Deficiency Conjoint with Marginal Vitamin C Deficiency Causes Cigarette Smoke Induced Myelodysplastic Syndromes

    doi: 10.1371/journal.pone.0020590

    Figure Lengend Snippet: MDS produced in the guinea pigs are irreversible. (Panel A ) Differential staining showing persistent changes in blood and bone marrow cell morphology of MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. A, a–d , represent sham controls (fed 0.5 mg vitamin C/day and exposed to air); A, e–h , represent MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day. Blood smear - Leishman stain; bone marrow aspirate - Wright Geimsa stain, except Perls' stain in d and h ; (magnification 400×). (Panel B ) Measurement of CD34(+) cells in bone marrow by flow cytometry. (Panel C ) Geimsa-stained metaphase spread showing aneuploidy in MDS guinea pigs followed by discontinuation of CS exposure and feeding 15 mg vitamin C/day; (magnification 1000×). (Panel D ) NQO1 Activity of bone marrow cells. Bars over the respective columns represent means ± SD (n = 4); * indicates significant difference (p

    Article Snippet: Supporting Information NQO1 expression in the bone marrow cells, indicating t hat there is no significant change in NQO1 expression at the protein level. (Panel A) and (Panel B) Bone marrow lysates were separated by 10% SDS PAGE and subjected to immmunobloting using antibody against NQO1.

    Techniques: Produced, Staining, Leishman Stain, Flow Cytometry, Cytometry, Activity Assay

    Immunofluorescent staining of NQO1 protein in BxPc-3 cells. NQO1 protein located in the cytoplasm and nucleus of BxPc-3 cells (green indicates NQO1; blue indicates DAPI; original magnification, ×630). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1.

    Journal: Oncology Letters

    Article Title: Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma

    doi: 10.3892/ol.2017.5821

    Figure Lengend Snippet: Immunofluorescent staining of NQO1 protein in BxPc-3 cells. NQO1 protein located in the cytoplasm and nucleus of BxPc-3 cells (green indicates NQO1; blue indicates DAPI; original magnification, ×630). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1.

    Article Snippet: The cells were subsequently incubated with anti-NQO1 antibody (dilution, 1:200; cat. no. A180:sc-32793; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight.

    Techniques: Staining

    Immunohistochemical staining of NQO1 protein expression in PDAC tissues. (A) NQO1 protein expression was evaluated in microarray of PDAC tissues. (B) NQO1 staining demonstrated negative expression in non-tumor tissues. (C) Strongly positive expression of NQO1 protein in PDAC with metastasis. (D) NQO1 protein was weakly positive expressed in PDAC. (E) NQO1 protein was negatively expressed in PDAC (Original magnification, ×200 in B-E). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1; PDAC, pancreatic ductal adenocarcinoma.

    Journal: Oncology Letters

    Article Title: Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma

    doi: 10.3892/ol.2017.5821

    Figure Lengend Snippet: Immunohistochemical staining of NQO1 protein expression in PDAC tissues. (A) NQO1 protein expression was evaluated in microarray of PDAC tissues. (B) NQO1 staining demonstrated negative expression in non-tumor tissues. (C) Strongly positive expression of NQO1 protein in PDAC with metastasis. (D) NQO1 protein was weakly positive expressed in PDAC. (E) NQO1 protein was negatively expressed in PDAC (Original magnification, ×200 in B-E). NQO1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1; PDAC, pancreatic ductal adenocarcinoma.

    Article Snippet: The cells were subsequently incubated with anti-NQO1 antibody (dilution, 1:200; cat. no. A180:sc-32793; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight.

    Techniques: Immunohistochemistry, Staining, Expressing, Microarray

    Kaplan-Meier survival curves of PDAC patients in early and late stage. (A) Kaplan-Meier curves for OS in early-stage PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P

    Journal: Oncology Letters

    Article Title: Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma

    doi: 10.3892/ol.2017.5821

    Figure Lengend Snippet: Kaplan-Meier survival curves of PDAC patients in early and late stage. (A) Kaplan-Meier curves for OS in early-stage PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P

    Article Snippet: The cells were subsequently incubated with anti-NQO1 antibody (dilution, 1:200; cat. no. A180:sc-32793; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight.

    Techniques: Expressing

    Kaplan-Meier survival curves illustrating the significance of NQO1 expression in PDAC patients with different grading. (A) OS rates of patients with high (solid; n=83) and low (dashed; n=43) NQO1 expression (P

    Journal: Oncology Letters

    Article Title: Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma

    doi: 10.3892/ol.2017.5821

    Figure Lengend Snippet: Kaplan-Meier survival curves illustrating the significance of NQO1 expression in PDAC patients with different grading. (A) OS rates of patients with high (solid; n=83) and low (dashed; n=43) NQO1 expression (P

    Article Snippet: The cells were subsequently incubated with anti-NQO1 antibody (dilution, 1:200; cat. no. A180:sc-32793; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight.

    Techniques: Expressing

    Association between NQO1 expression and clinicopathological significance of PDAC. The expression level of NQO1 protein was significantly associated with (A) grading (P

    Journal: Oncology Letters

    Article Title: Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma

    doi: 10.3892/ol.2017.5821

    Figure Lengend Snippet: Association between NQO1 expression and clinicopathological significance of PDAC. The expression level of NQO1 protein was significantly associated with (A) grading (P

    Article Snippet: The cells were subsequently incubated with anti-NQO1 antibody (dilution, 1:200; cat. no. A180:sc-32793; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight.

    Techniques: Expressing

    Kaplan-Meier survival curves of PDAC patients with lymph node metastasis and without metastasis. (A) Kaplan-Meier curves for OS in the absence of LN metastasis in PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P

    Journal: Oncology Letters

    Article Title: Clinicopathological implications of NQO1 overexpression in the prognosis of pancreatic adenocarcinoma

    doi: 10.3892/ol.2017.5821

    Figure Lengend Snippet: Kaplan-Meier survival curves of PDAC patients with lymph node metastasis and without metastasis. (A) Kaplan-Meier curves for OS in the absence of LN metastasis in PDAC patients with low and high level of NQO1 expression (log-rank=18.402, P

    Article Snippet: The cells were subsequently incubated with anti-NQO1 antibody (dilution, 1:200; cat. no. A180:sc-32793; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight.

    Techniques: Expressing

    NQO1 inhibits PGC-1α proteasomal degradation and increases its protein half-life. (A) HEK-293T cells and HEK-293T cells stably expressing the Flag-tagged β4 (PSMB2) ring subunit were transiently transfected with untagged PGC-1α-

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: NQO1 inhibits PGC-1α proteasomal degradation and increases its protein half-life. (A) HEK-293T cells and HEK-293T cells stably expressing the Flag-tagged β4 (PSMB2) ring subunit were transiently transfected with untagged PGC-1α-

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Pyrolysis Gas Chromatography, Stable Transfection, Expressing, Transfection

    NQO1 knockdown in myoblasts reduces steady-state PGC-1α protein levels and activity. (A) NQO1 was knocked down in C2C12 cells using a lentivirus-based approach (3 individual experiments); all were run on the same gel, and cells were analyzed for

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: NQO1 knockdown in myoblasts reduces steady-state PGC-1α protein levels and activity. (A) NQO1 was knocked down in C2C12 cells using a lentivirus-based approach (3 individual experiments); all were run on the same gel, and cells were analyzed for

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Pyrolysis Gas Chromatography, Activity Assay

    NQO1 protects PGC-1α by NADH-dependent interaction. (A) HEK-293 cells were transfected as indicated and harvested for analysis 24 h posttransfection. HA beads were used to immunoprecipitate (IP) HA-tagged PGC-1α, whereas HA-p73β

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: NQO1 protects PGC-1α by NADH-dependent interaction. (A) HEK-293 cells were transfected as indicated and harvested for analysis 24 h posttransfection. HA beads were used to immunoprecipitate (IP) HA-tagged PGC-1α, whereas HA-p73β

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Pyrolysis Gas Chromatography, Transfection

    PGC-1α is an intrinsically disordered protein by prediction, susceptible to in vitro degradation by the 20S PC and protected by NQO1. (A) Analysis of PGC-1α and PCNA amino acid sequences by the FoldIndex prediction program. (B) In vitro

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: PGC-1α is an intrinsically disordered protein by prediction, susceptible to in vitro degradation by the 20S PC and protected by NQO1. (A) Analysis of PGC-1α and PCNA amino acid sequences by the FoldIndex prediction program. (B) In vitro

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Pyrolysis Gas Chromatography, In Vitro

    Schematic model illustrating the convergent actions of CREB, NQO1, AMPK, and Sirt1 on PGC-1α. The scheme summarizes PGC-1α transcription and posttranslational regulation by different known metabolite-sensing proteins, including NQO1.

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: Schematic model illustrating the convergent actions of CREB, NQO1, AMPK, and Sirt1 on PGC-1α. The scheme summarizes PGC-1α transcription and posttranslational regulation by different known metabolite-sensing proteins, including NQO1.

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Pyrolysis Gas Chromatography

    A role for NQO1 activity in regulating PGC-1α in fasting liver. (A) Mice were either fed ad libitum or fasted during the indicated time points (starting from the beginning of the night phase) and then sacrificed for hepatic analysis of NADH and

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: A role for NQO1 activity in regulating PGC-1α in fasting liver. (A) Mice were either fed ad libitum or fasted during the indicated time points (starting from the beginning of the night phase) and then sacrificed for hepatic analysis of NADH and

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Activity Assay, Pyrolysis Gas Chromatography, Mouse Assay

    NQO1 and PGC-1α steady-state levels in myoblasts and myotubes. (A) Analysis of PGC-1α and NQO1 protein and mRNA levels in C2C12 myoblast (MB) cells and at sequential days after the cells were put in differentiation medium. (B) Same as

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: NQO1 and PGC-1α steady-state levels in myoblasts and myotubes. (A) Analysis of PGC-1α and NQO1 protein and mRNA levels in C2C12 myoblast (MB) cells and at sequential days after the cells were put in differentiation medium. (B) Same as

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Pyrolysis Gas Chromatography

    NQO1 regulates PGC-1α accumulation in response to induction by starvation-mimicking conditions in mouse primary hepatocytes. (A) Primary hepatocytes were infected with either GFP- or NQO1-expressing adenoviruses. Expression levels of NQO1 mRNA

    Journal: Molecular and Cellular Biology

    Article Title: The Protein Level of PGC-1?, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

    doi: 10.1128/MCB.01672-12

    Figure Lengend Snippet: NQO1 regulates PGC-1α accumulation in response to induction by starvation-mimicking conditions in mouse primary hepatocytes. (A) Primary hepatocytes were infected with either GFP- or NQO1-expressing adenoviruses. Expression levels of NQO1 mRNA

    Article Snippet: The antibodies used were as follows: monoclonal mouse antiactin, anti-HA, anti-Flag (Sigma), mouse anti-NQO1 (Santa Cruz Biotechnology), mouse anti-PGC-1α (Calbiochem), polyclonal goat anti-NQO1 (Santa Cruz Biotechnology), polyclonal goat anti-NQO1 (ab2346; Abcam), and polyclonal rabbit anti-acetylated-Lys (9441; Cell Signaling).

    Techniques: Pyrolysis Gas Chromatography, Infection, Expressing