anti-nqo1 Search Results


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  • 90
    Thermo Fisher anti nqo1
    <t>NQO1</t> regulates HIF-1α stability in cancer cells. ( a , b ) NQO1 increases the stability of HIF-1α protein under normoxia. Cells were transfected with pHIF-1α-myc-His 6 and incubated with 10 μg ml −1 cycloheximide for 3 min, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( a ) and MDA-MB-231 ( b ) cells. β-actin was used as the internal control. ( c , d ) NQO1 prolongs the half-life of HIF-1α protein under hypoxia. Cells were transfected with pHIF-1α-myc-His 6 , exposed to 0.5% O 2 for 2 h, incubated with 10 μg ml −1 cycloheximide for 2 h, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( c ) and MDA-MB-231 ( d ) cells. β-actin was used as the internal control. HIF-1α protein levels were quantified using Image J, and band intensities were normalized to those of β-actin (band intensity at t 0 was defined as 100%).
    Anti Nqo1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam anti nqo 1
    Expression of Robo family members in the olfactory epithelium. In situ hybridization of coronal sections of olfactory epithelium isolated from E16 embryos ( A–E ) and adult mice (P90) ( F–M ) with cRNA probes specific for robo-1 ( A ), rig-1 ( B ), robo-2 ( C , G , H ), <t>NQO-1</t> ( D , I ), OCAM ( E , J ), OMP ( F ), M49 ( K ), L45 ( L ), and M50 ( M ). A–G , Expression of robo-1 is restricted to the basal lamina of the olfactory epithelium ( A , arrowheads), whereas rig-1 is not expressed in the olfactory epithelium ( B ). Interestingly, in contrast to OMP, which is equally expressed throughout the olfactory epithelium, robo-2 is expressed in a gradient with high levels of expression in the dorsomedial regions of the olfactory epithelium (arrows) and low levels in the ventrolateral regions (arrowheads) of E16 ( C ) and adult olfactory epithelium ( G , H ). H–M , robo-2 expression is confined to zones I–III of the olfactory epithelium. A higher magnification of a region of the olfactory epithelium (boxed in G ) in which all four zones of the olfactory epithelium are represented is shown in H–M . The expression pattern of robo-2 was compared with the expression pattern of specific olfactory epithelium zonal markers that include NQO-1 (zone I) ( I ), OCAM (zones II–IV) ( J ), M49 (zone II) ( K ), L45 (zone III) ( L ), and M50 (zone IV) ( M ). robo-2 is expressed in a high-to-low gradient in olfactory sensory neurons located in zones I (arrows) to IV (arrowheads), respectively. Regions of the olfactory epithelium expressing the different zonal markers are traced with a colored line on the apical surface of the olfactory epithelium to represent the four zones (zone I, magenta; zone II, green; zone III, yellow; zone IV, blue). S, Septum. Scale bars: A–E , H–M , 250 μm; F , G , 500 μm. N , Diagram representing the spatial relationship between the location of olfactory sensory neurons within the OE and their target glomeruli within the OB. Olfactory sensory neurons located in the dorsomedial regions of the olfactory epithelium (magenta) project axons to glomeruli in the dorsal region of the olfactory bulb, whereas olfactory sensory neurons in the ventrolateral region of the olfactory epithelium (blue and not shown) project axons to the ventral aspect of the olfactory bulb. D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal.
    Anti Nqo 1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam mouse anti nqo 1
    Nrf2-activators dose-dependently increase antioxidant protein expression in OLN-93 cells. HO-1 ( A ), <t>NQO-1</t> ( B ) and p62 ( C ) protein expression levels after 24 h treatment in the OLN-93 oligodendrocyte cell line with 5 µM or 10 µM SFN, 45 µM or 90 µM MMF, 30 µg/mL or 60 µg/mL Protandim or their respective DMSO or EtOH vehicle control. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. All statistics reflect one-way ANOVA tests with post hoc Bonferroni correction; * p
    Mouse Anti Nqo 1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher anti nqo1 antibody
    β-Lap causes cell death and increases cellular radiosensitivity in <t>NQO1</t> dependent manner. (A) Effects of β-lap on the apoptosis in wild type A549 cells and shQO1 A549 cells. (B) Effects of β-lap on the clonogenic survival of wild type A549 cells and shNQO1 A549 cells. An average of seven experiments ± SEM is shown.
    Anti Nqo1 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc anti nqo1
    Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, <t>NQO1</t> and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P
    Anti Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Proteintech nqo1
    Western blot analysis of <t>NQO1,</t> γ-GCS, and HO-1 levels in the lungs of bleomycin-induced pulmonary fibrosis rats. The decreased levels of NQO1, γ-GCS, and HO-1 protein expression were significantly increased by administration of BMSCs.
    Nqo1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech anti nqo1
    Western blot analysis of <t>NQO1,</t> γ-GCS, and HO-1 levels in the lungs of bleomycin-induced pulmonary fibrosis rats. The decreased levels of NQO1, γ-GCS, and HO-1 protein expression were significantly increased by administration of BMSCs.
    Anti Nqo1, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare anti nqo1
    <t>NQO1</t> regulates HIF-1α stability in cancer cells. ( a , b ) NQO1 increases the stability of HIF-1α protein under normoxia. Cells were transfected with pHIF-1α-myc-His 6 and incubated with 10 μg ml −1 cycloheximide for 3 min, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( a ) and MDA-MB-231 ( b ) cells. β-actin was used as the internal control. ( c , d ) NQO1 prolongs the half-life of HIF-1α protein under hypoxia. Cells were transfected with pHIF-1α-myc-His 6 , exposed to 0.5% O 2 for 2 h, incubated with 10 μg ml −1 cycloheximide for 2 h, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( c ) and MDA-MB-231 ( d ) cells. β-actin was used as the internal control. HIF-1α protein levels were quantified using Image J, and band intensities were normalized to those of β-actin (band intensity at t 0 was defined as 100%).
    Anti Nqo1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nqo1  (Abnova)
    91
    Abnova nqo1
    Accumulation of Nrf2 in the nuclear fraction in df-SH-SY5Y cells. (A) Cells were treated with or without 200 μM Na 2 S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na 2 S) for 2 h, and Nrf2 protein levels in nuclear fractions were analyzed by western blotting. Lamin B1 was used as nuclear loading control (bottom panel). (B) Cells were treated with or without 200 μM Na 2 S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na 2 S) for 10 h, and <t>NQO1</t> protein levels in cytosol fractions were analyzed by western blotting. β-actin was used as cytosol loading control (bottom panel). (C) Cells were treated with 0, 100 and 200 μM Na 2 S or BS-Mix (prepared by 200 μM Na 2 S) for 2 h, and GSH levels were measured using HPLC-FL with ABD-F derivatized samples. Values indicate means±S.D. * P
    Nqo1, supplied by Abnova, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epitomics anti nqo1
    Western blot analysis of Nrf2 target proteins. A, After 28 days of diet, Western blot of 3 random animals from each experimental group was performed for the prototypical Nrf2-dependent proteins: GCLM, GstA2, and <t>Nqo1.</t> B, Densitometry of the presented blot demonstrated significant increases in the examined Nrf2-dependent proteins in the AlbCre+/caNrf2+ animal when compared with AlbCre+/caNrf2− animals independent of diet. * P
    Anti Nqo1, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Boster Bio nqo1
    Linagliptin activated the NRF2 antioxidant pathway. The expression levels of the KEAP1, nuclear NRF2, HO-1 and <t>NQO1</t> were determined by Western blot (A). The binding capacity of NRF2 to ARE was assessed by EMSA (B).The data were presented as mean ± SD (n=6 for each group. *P
    Nqo1, supplied by Boster Bio, used in various techniques. Bioz Stars score: 91/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    NQO1 regulates HIF-1α stability in cancer cells. ( a , b ) NQO1 increases the stability of HIF-1α protein under normoxia. Cells were transfected with pHIF-1α-myc-His 6 and incubated with 10 μg ml −1 cycloheximide for 3 min, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( a ) and MDA-MB-231 ( b ) cells. β-actin was used as the internal control. ( c , d ) NQO1 prolongs the half-life of HIF-1α protein under hypoxia. Cells were transfected with pHIF-1α-myc-His 6 , exposed to 0.5% O 2 for 2 h, incubated with 10 μg ml −1 cycloheximide for 2 h, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( c ) and MDA-MB-231 ( d ) cells. β-actin was used as the internal control. HIF-1α protein levels were quantified using Image J, and band intensities were normalized to those of β-actin (band intensity at t 0 was defined as 100%).

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 regulates HIF-1α stability in cancer cells. ( a , b ) NQO1 increases the stability of HIF-1α protein under normoxia. Cells were transfected with pHIF-1α-myc-His 6 and incubated with 10 μg ml −1 cycloheximide for 3 min, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( a ) and MDA-MB-231 ( b ) cells. β-actin was used as the internal control. ( c , d ) NQO1 prolongs the half-life of HIF-1α protein under hypoxia. Cells were transfected with pHIF-1α-myc-His 6 , exposed to 0.5% O 2 for 2 h, incubated with 10 μg ml −1 cycloheximide for 2 h, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( c ) and MDA-MB-231 ( d ) cells. β-actin was used as the internal control. HIF-1α protein levels were quantified using Image J, and band intensities were normalized to those of β-actin (band intensity at t 0 was defined as 100%).

    Article Snippet: Antibodies were obtained from the following sources: Anti-HIF-1α (1:500; MAB1536, R & D Systems), anti-PHD1 (1:1,000; AF6394, R & D Systems), anti-Hydroxylated-HIF-1α (1:1,000; #5853S, Cell Signaling Technology), anti- HIF-1β (1:1,000; #3414S, Cell Signaling Technology), anti-PHD2 (1:1,000; #3293, Cell Signaling Technology), anti-RACK1 (1:1,000; #5432S, Cell Signaling Technology), anti-Ki67 (1:200; #12202, Cell Signaling Technology), anti-CC3 (1:200; #9664, Cell Signaling Technology), anti-PHD3 (1:1,000; NB-100-139, NOVUS), anti-NQO1 (1:2,000; 39–3,700, Invitrogen), anti-ubiquitin (1:500; 131,600, Invitrogen), anti-β-actin (1:5,000; A5316, Sigma-Aldrich), anti-Cullin5 (1:1,000; sc-13014, Santa Cruz Biotechnology), anti-HSP70 (1:2,000; ADI-SPA-820D, Stratagene), anti-HSP90 (1:2,000; ADI-SPA-830D, Stratagene), anti-His6 (1:1,000; 11 911 416 001, Roche Applied Science), anti- GFP (1:1,000; 11 814 460 001, Roche Applied Science), anti-CD34 (1:200; ab8158, Abcam), anti-pimonidazole (1:200; HP6-x, Hydroxyprobe).

    Techniques: Transfection, Incubation, Multiple Displacement Amplification

    NQO1 enhances HIF-1α expression. ( a , b ) RKO cells ( a ) and MDA-MB-231 cells ( b ) were exposed to 0.5% O 2 for 8 h and harvested at the indicated times. Whole-cell lysates were analysed by immunoblotting for HIF-1α, HIF-1β, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( c , d ) Various concentrations of pshNQO1 and pNQO1-myc-His 6 (0–10 μg) were transiently transfected into RKO ( c ) and MDA-MB-231 ( d ) cells, respectively. After 48 h, the cells were exposed to 20 or 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. EV indicates pshCont ( c ) and pCDNA3.1-myc-His 6 ( d ). SE, short exposure; LE, long exposure. ( e ) pNQO1-myc-His 6 was transiently transfected into RKO/pshNQO1 cells. After 48 h, the cells were exposed to 0.5% O 2 for 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( f ) Mouse embryonic fibroblast wild type and NQO1 −/− cells were exposed to 20 or 0.5% O 2 for 2 h and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure.

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 enhances HIF-1α expression. ( a , b ) RKO cells ( a ) and MDA-MB-231 cells ( b ) were exposed to 0.5% O 2 for 8 h and harvested at the indicated times. Whole-cell lysates were analysed by immunoblotting for HIF-1α, HIF-1β, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( c , d ) Various concentrations of pshNQO1 and pNQO1-myc-His 6 (0–10 μg) were transiently transfected into RKO ( c ) and MDA-MB-231 ( d ) cells, respectively. After 48 h, the cells were exposed to 20 or 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. EV indicates pshCont ( c ) and pCDNA3.1-myc-His 6 ( d ). SE, short exposure; LE, long exposure. ( e ) pNQO1-myc-His 6 was transiently transfected into RKO/pshNQO1 cells. After 48 h, the cells were exposed to 0.5% O 2 for 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( f ) Mouse embryonic fibroblast wild type and NQO1 −/− cells were exposed to 20 or 0.5% O 2 for 2 h and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure.

    Article Snippet: Antibodies were obtained from the following sources: Anti-HIF-1α (1:500; MAB1536, R & D Systems), anti-PHD1 (1:1,000; AF6394, R & D Systems), anti-Hydroxylated-HIF-1α (1:1,000; #5853S, Cell Signaling Technology), anti- HIF-1β (1:1,000; #3414S, Cell Signaling Technology), anti-PHD2 (1:1,000; #3293, Cell Signaling Technology), anti-RACK1 (1:1,000; #5432S, Cell Signaling Technology), anti-Ki67 (1:200; #12202, Cell Signaling Technology), anti-CC3 (1:200; #9664, Cell Signaling Technology), anti-PHD3 (1:1,000; NB-100-139, NOVUS), anti-NQO1 (1:2,000; 39–3,700, Invitrogen), anti-ubiquitin (1:500; 131,600, Invitrogen), anti-β-actin (1:5,000; A5316, Sigma-Aldrich), anti-Cullin5 (1:1,000; sc-13014, Santa Cruz Biotechnology), anti-HSP70 (1:2,000; ADI-SPA-820D, Stratagene), anti-HSP90 (1:2,000; ADI-SPA-830D, Stratagene), anti-His6 (1:1,000; 11 911 416 001, Roche Applied Science), anti- GFP (1:1,000; 11 814 460 001, Roche Applied Science), anti-CD34 (1:200; ab8158, Abcam), anti-pimonidazole (1:200; HP6-x, Hydroxyprobe).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection

    NQO1 stabilizes HIF-1α through inhibiting ubiquitination and proteasome-mediated degradation. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively. The cells were then exposed to 0.5% O 2 for 2 h, and incubated for 1 h in the presence or absence of MG132. Whole-cell extracts were immunoprecipitated with an anti- HIF-1α antibody, and ubiquitinated HIF-1α was detected with an anti-ubiquitin antibody. ( c , d ) RKO ( c ) and MDA-MB-231 ( d ) cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were treated with or without cycloheximide and MG132, incubated for 1 h, and harvested. The collected cells were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. ( e , f ) RKO cells ( e ) and MDA-MB-231 cells ( f ) were exposed to 0.5% O 2 . After 2 h, the cells were harvested and immunoprecipitated with anti- HIF-1α or anti-IgG (negative control) antibodies. The precipitates were analysed by immunoblotting with the indicated antibodies. ( g ) RKO/pshCont and RKO/pshNQO1 cells were treated with or without 1 mM of DMOG for 1 h, and then exposed to 0.5% O 2 . After 2 h, the cells were harvested and analysed by immunoblotting with the indicated antibodies. ( h ) RKO/pshCont and RKO/pshNQO1 cells were transfected with siPHD1, siPHD2, siPHD3 or HIF-1α. After 48 h, the cells were treated with 50 μg MG132 for 6 h, exposed to 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, hydroxylated-HIF-1α, PHD1, PHD2, PHD3, NQO1 and β-actin. X-ray films were exposed for 1, 3 or 5 min to detect the signals of total HIF-1α, hydroxylated-HIF-1α.

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 stabilizes HIF-1α through inhibiting ubiquitination and proteasome-mediated degradation. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively. The cells were then exposed to 0.5% O 2 for 2 h, and incubated for 1 h in the presence or absence of MG132. Whole-cell extracts were immunoprecipitated with an anti- HIF-1α antibody, and ubiquitinated HIF-1α was detected with an anti-ubiquitin antibody. ( c , d ) RKO ( c ) and MDA-MB-231 ( d ) cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were treated with or without cycloheximide and MG132, incubated for 1 h, and harvested. The collected cells were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. ( e , f ) RKO cells ( e ) and MDA-MB-231 cells ( f ) were exposed to 0.5% O 2 . After 2 h, the cells were harvested and immunoprecipitated with anti- HIF-1α or anti-IgG (negative control) antibodies. The precipitates were analysed by immunoblotting with the indicated antibodies. ( g ) RKO/pshCont and RKO/pshNQO1 cells were treated with or without 1 mM of DMOG for 1 h, and then exposed to 0.5% O 2 . After 2 h, the cells were harvested and analysed by immunoblotting with the indicated antibodies. ( h ) RKO/pshCont and RKO/pshNQO1 cells were transfected with siPHD1, siPHD2, siPHD3 or HIF-1α. After 48 h, the cells were treated with 50 μg MG132 for 6 h, exposed to 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, hydroxylated-HIF-1α, PHD1, PHD2, PHD3, NQO1 and β-actin. X-ray films were exposed for 1, 3 or 5 min to detect the signals of total HIF-1α, hydroxylated-HIF-1α.

    Article Snippet: Antibodies were obtained from the following sources: Anti-HIF-1α (1:500; MAB1536, R & D Systems), anti-PHD1 (1:1,000; AF6394, R & D Systems), anti-Hydroxylated-HIF-1α (1:1,000; #5853S, Cell Signaling Technology), anti- HIF-1β (1:1,000; #3414S, Cell Signaling Technology), anti-PHD2 (1:1,000; #3293, Cell Signaling Technology), anti-RACK1 (1:1,000; #5432S, Cell Signaling Technology), anti-Ki67 (1:200; #12202, Cell Signaling Technology), anti-CC3 (1:200; #9664, Cell Signaling Technology), anti-PHD3 (1:1,000; NB-100-139, NOVUS), anti-NQO1 (1:2,000; 39–3,700, Invitrogen), anti-ubiquitin (1:500; 131,600, Invitrogen), anti-β-actin (1:5,000; A5316, Sigma-Aldrich), anti-Cullin5 (1:1,000; sc-13014, Santa Cruz Biotechnology), anti-HSP70 (1:2,000; ADI-SPA-820D, Stratagene), anti-HSP90 (1:2,000; ADI-SPA-830D, Stratagene), anti-His6 (1:1,000; 11 911 416 001, Roche Applied Science), anti- GFP (1:1,000; 11 814 460 001, Roche Applied Science), anti-CD34 (1:200; ab8158, Abcam), anti-pimonidazole (1:200; HP6-x, Hydroxyprobe).

    Techniques: Multiple Displacement Amplification, Transfection, Incubation, Immunoprecipitation, Negative Control

    NQO1 interacts with HIF-1α in cytosol. ( a ) RKO cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were harvested and lysates were co-immunoprecipitated with anti-HIF-1α (left) or anti-NQO1 (right) antibody and anti-IgG antibody as a negative control. Then, the precipitates were analysed by immunoblot analysis with anti -HIF-1α and -NQO1 antibodies. ( b ) RKO cells were transfected with HIF-1α-myc-His 6 (left) or NQO1-myc-His 6 (right), subjected to Ni-NTA bead-based pulldown, and analysed by immunoblotting with anti-His 6 , -HIF-1α and -NQO1 antibodies. ( c ) Illustration of HIF-1α domain. ( d , e ) GFP pulldown assays were performed with purified EGFP fusion proteins containing the indicated amino-acid residues of HIF-1α, along with whole-cell lysates from RKO cells. The immunoprecipitated proteins were analysed by immunoblotting with anti-GFP and -NQO1 antibodies. ( f ) Extracted cytosolic or nuclear protein was immunoprecipitated with anti- HIF-1α (upper panel) or anti-NQO1 (lower panel) antibodies and analysed by immunoblotting to detect HIF-1α and NQO1.

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 interacts with HIF-1α in cytosol. ( a ) RKO cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were harvested and lysates were co-immunoprecipitated with anti-HIF-1α (left) or anti-NQO1 (right) antibody and anti-IgG antibody as a negative control. Then, the precipitates were analysed by immunoblot analysis with anti -HIF-1α and -NQO1 antibodies. ( b ) RKO cells were transfected with HIF-1α-myc-His 6 (left) or NQO1-myc-His 6 (right), subjected to Ni-NTA bead-based pulldown, and analysed by immunoblotting with anti-His 6 , -HIF-1α and -NQO1 antibodies. ( c ) Illustration of HIF-1α domain. ( d , e ) GFP pulldown assays were performed with purified EGFP fusion proteins containing the indicated amino-acid residues of HIF-1α, along with whole-cell lysates from RKO cells. The immunoprecipitated proteins were analysed by immunoblotting with anti-GFP and -NQO1 antibodies. ( f ) Extracted cytosolic or nuclear protein was immunoprecipitated with anti- HIF-1α (upper panel) or anti-NQO1 (lower panel) antibodies and analysed by immunoblotting to detect HIF-1α and NQO1.

    Article Snippet: Antibodies were obtained from the following sources: Anti-HIF-1α (1:500; MAB1536, R & D Systems), anti-PHD1 (1:1,000; AF6394, R & D Systems), anti-Hydroxylated-HIF-1α (1:1,000; #5853S, Cell Signaling Technology), anti- HIF-1β (1:1,000; #3414S, Cell Signaling Technology), anti-PHD2 (1:1,000; #3293, Cell Signaling Technology), anti-RACK1 (1:1,000; #5432S, Cell Signaling Technology), anti-Ki67 (1:200; #12202, Cell Signaling Technology), anti-CC3 (1:200; #9664, Cell Signaling Technology), anti-PHD3 (1:1,000; NB-100-139, NOVUS), anti-NQO1 (1:2,000; 39–3,700, Invitrogen), anti-ubiquitin (1:500; 131,600, Invitrogen), anti-β-actin (1:5,000; A5316, Sigma-Aldrich), anti-Cullin5 (1:1,000; sc-13014, Santa Cruz Biotechnology), anti-HSP70 (1:2,000; ADI-SPA-820D, Stratagene), anti-HSP90 (1:2,000; ADI-SPA-830D, Stratagene), anti-His6 (1:1,000; 11 911 416 001, Roche Applied Science), anti- GFP (1:1,000; 11 814 460 001, Roche Applied Science), anti-CD34 (1:200; ab8158, Abcam), anti-pimonidazole (1:200; HP6-x, Hydroxyprobe).

    Techniques: Immunoprecipitation, Negative Control, Transfection, Purification

    NQO1 increases transcriptional activity of HIF-1α in cancer cells under hypoxia. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively, exposed to 20 or 0.5% O 2 for 8 h, and then harvested. Quantitative PCR was used to amplify PDK1, PGK1 and LDHA. The signals were normalized by 18S rRNA . (mean±s.d. shown) n =3 in each group. * P

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 increases transcriptional activity of HIF-1α in cancer cells under hypoxia. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively, exposed to 20 or 0.5% O 2 for 8 h, and then harvested. Quantitative PCR was used to amplify PDK1, PGK1 and LDHA. The signals were normalized by 18S rRNA . (mean±s.d. shown) n =3 in each group. * P

    Article Snippet: Antibodies were obtained from the following sources: Anti-HIF-1α (1:500; MAB1536, R & D Systems), anti-PHD1 (1:1,000; AF6394, R & D Systems), anti-Hydroxylated-HIF-1α (1:1,000; #5853S, Cell Signaling Technology), anti- HIF-1β (1:1,000; #3414S, Cell Signaling Technology), anti-PHD2 (1:1,000; #3293, Cell Signaling Technology), anti-RACK1 (1:1,000; #5432S, Cell Signaling Technology), anti-Ki67 (1:200; #12202, Cell Signaling Technology), anti-CC3 (1:200; #9664, Cell Signaling Technology), anti-PHD3 (1:1,000; NB-100-139, NOVUS), anti-NQO1 (1:2,000; 39–3,700, Invitrogen), anti-ubiquitin (1:500; 131,600, Invitrogen), anti-β-actin (1:5,000; A5316, Sigma-Aldrich), anti-Cullin5 (1:1,000; sc-13014, Santa Cruz Biotechnology), anti-HSP70 (1:2,000; ADI-SPA-820D, Stratagene), anti-HSP90 (1:2,000; ADI-SPA-830D, Stratagene), anti-His6 (1:1,000; 11 911 416 001, Roche Applied Science), anti- GFP (1:1,000; 11 814 460 001, Roche Applied Science), anti-CD34 (1:200; ab8158, Abcam), anti-pimonidazole (1:200; HP6-x, Hydroxyprobe).

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction

    Upregulation of NQO1 correlates with poor prognosis and expression of HIF-1α in colorectal cancer. ( a ) An Oncomine analysis of the TCGA colorectal database indicated that NQO1 expressions are elevated in colorectal cancers ( n =102) compared with normal colorectal tissues ( n =19). **** P

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: Upregulation of NQO1 correlates with poor prognosis and expression of HIF-1α in colorectal cancer. ( a ) An Oncomine analysis of the TCGA colorectal database indicated that NQO1 expressions are elevated in colorectal cancers ( n =102) compared with normal colorectal tissues ( n =19). **** P

    Article Snippet: Antibodies were obtained from the following sources: Anti-HIF-1α (1:500; MAB1536, R & D Systems), anti-PHD1 (1:1,000; AF6394, R & D Systems), anti-Hydroxylated-HIF-1α (1:1,000; #5853S, Cell Signaling Technology), anti- HIF-1β (1:1,000; #3414S, Cell Signaling Technology), anti-PHD2 (1:1,000; #3293, Cell Signaling Technology), anti-RACK1 (1:1,000; #5432S, Cell Signaling Technology), anti-Ki67 (1:200; #12202, Cell Signaling Technology), anti-CC3 (1:200; #9664, Cell Signaling Technology), anti-PHD3 (1:1,000; NB-100-139, NOVUS), anti-NQO1 (1:2,000; 39–3,700, Invitrogen), anti-ubiquitin (1:500; 131,600, Invitrogen), anti-β-actin (1:5,000; A5316, Sigma-Aldrich), anti-Cullin5 (1:1,000; sc-13014, Santa Cruz Biotechnology), anti-HSP70 (1:2,000; ADI-SPA-820D, Stratagene), anti-HSP90 (1:2,000; ADI-SPA-830D, Stratagene), anti-His6 (1:1,000; 11 911 416 001, Roche Applied Science), anti- GFP (1:1,000; 11 814 460 001, Roche Applied Science), anti-CD34 (1:200; ab8158, Abcam), anti-pimonidazole (1:200; HP6-x, Hydroxyprobe).

    Techniques: Expressing

    NQO1 promotes in vivo tumour growth. ( a ) RKO/pNQO1, RKO/shCont and RKO/pshNQO1 cells were injected subcutaneously into the right flank of athymic, 7-week-old female BALB/C nude mice, and tumour growth was assessed. Tumour volume (TV) was calculated by using the following formula: TV=length × (width) 2 × 0.5. Each group contained 12 animals. (** P

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 promotes in vivo tumour growth. ( a ) RKO/pNQO1, RKO/shCont and RKO/pshNQO1 cells were injected subcutaneously into the right flank of athymic, 7-week-old female BALB/C nude mice, and tumour growth was assessed. Tumour volume (TV) was calculated by using the following formula: TV=length × (width) 2 × 0.5. Each group contained 12 animals. (** P

    Article Snippet: Antibodies were obtained from the following sources: Anti-HIF-1α (1:500; MAB1536, R & D Systems), anti-PHD1 (1:1,000; AF6394, R & D Systems), anti-Hydroxylated-HIF-1α (1:1,000; #5853S, Cell Signaling Technology), anti- HIF-1β (1:1,000; #3414S, Cell Signaling Technology), anti-PHD2 (1:1,000; #3293, Cell Signaling Technology), anti-RACK1 (1:1,000; #5432S, Cell Signaling Technology), anti-Ki67 (1:200; #12202, Cell Signaling Technology), anti-CC3 (1:200; #9664, Cell Signaling Technology), anti-PHD3 (1:1,000; NB-100-139, NOVUS), anti-NQO1 (1:2,000; 39–3,700, Invitrogen), anti-ubiquitin (1:500; 131,600, Invitrogen), anti-β-actin (1:5,000; A5316, Sigma-Aldrich), anti-Cullin5 (1:1,000; sc-13014, Santa Cruz Biotechnology), anti-HSP70 (1:2,000; ADI-SPA-820D, Stratagene), anti-HSP90 (1:2,000; ADI-SPA-830D, Stratagene), anti-His6 (1:1,000; 11 911 416 001, Roche Applied Science), anti- GFP (1:1,000; 11 814 460 001, Roche Applied Science), anti-CD34 (1:200; ab8158, Abcam), anti-pimonidazole (1:200; HP6-x, Hydroxyprobe).

    Techniques: In Vivo, Injection, Mouse Assay

    Expression of Robo family members in the olfactory epithelium. In situ hybridization of coronal sections of olfactory epithelium isolated from E16 embryos ( A–E ) and adult mice (P90) ( F–M ) with cRNA probes specific for robo-1 ( A ), rig-1 ( B ), robo-2 ( C , G , H ), NQO-1 ( D , I ), OCAM ( E , J ), OMP ( F ), M49 ( K ), L45 ( L ), and M50 ( M ). A–G , Expression of robo-1 is restricted to the basal lamina of the olfactory epithelium ( A , arrowheads), whereas rig-1 is not expressed in the olfactory epithelium ( B ). Interestingly, in contrast to OMP, which is equally expressed throughout the olfactory epithelium, robo-2 is expressed in a gradient with high levels of expression in the dorsomedial regions of the olfactory epithelium (arrows) and low levels in the ventrolateral regions (arrowheads) of E16 ( C ) and adult olfactory epithelium ( G , H ). H–M , robo-2 expression is confined to zones I–III of the olfactory epithelium. A higher magnification of a region of the olfactory epithelium (boxed in G ) in which all four zones of the olfactory epithelium are represented is shown in H–M . The expression pattern of robo-2 was compared with the expression pattern of specific olfactory epithelium zonal markers that include NQO-1 (zone I) ( I ), OCAM (zones II–IV) ( J ), M49 (zone II) ( K ), L45 (zone III) ( L ), and M50 (zone IV) ( M ). robo-2 is expressed in a high-to-low gradient in olfactory sensory neurons located in zones I (arrows) to IV (arrowheads), respectively. Regions of the olfactory epithelium expressing the different zonal markers are traced with a colored line on the apical surface of the olfactory epithelium to represent the four zones (zone I, magenta; zone II, green; zone III, yellow; zone IV, blue). S, Septum. Scale bars: A–E , H–M , 250 μm; F , G , 500 μm. N , Diagram representing the spatial relationship between the location of olfactory sensory neurons within the OE and their target glomeruli within the OB. Olfactory sensory neurons located in the dorsomedial regions of the olfactory epithelium (magenta) project axons to glomeruli in the dorsal region of the olfactory bulb, whereas olfactory sensory neurons in the ventrolateral region of the olfactory epithelium (blue and not shown) project axons to the ventral aspect of the olfactory bulb. D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal.

    Journal: The Journal of Neuroscience

    Article Title: Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

    doi: 10.1523/JNEUROSCI.2217-07.2007

    Figure Lengend Snippet: Expression of Robo family members in the olfactory epithelium. In situ hybridization of coronal sections of olfactory epithelium isolated from E16 embryos ( A–E ) and adult mice (P90) ( F–M ) with cRNA probes specific for robo-1 ( A ), rig-1 ( B ), robo-2 ( C , G , H ), NQO-1 ( D , I ), OCAM ( E , J ), OMP ( F ), M49 ( K ), L45 ( L ), and M50 ( M ). A–G , Expression of robo-1 is restricted to the basal lamina of the olfactory epithelium ( A , arrowheads), whereas rig-1 is not expressed in the olfactory epithelium ( B ). Interestingly, in contrast to OMP, which is equally expressed throughout the olfactory epithelium, robo-2 is expressed in a gradient with high levels of expression in the dorsomedial regions of the olfactory epithelium (arrows) and low levels in the ventrolateral regions (arrowheads) of E16 ( C ) and adult olfactory epithelium ( G , H ). H–M , robo-2 expression is confined to zones I–III of the olfactory epithelium. A higher magnification of a region of the olfactory epithelium (boxed in G ) in which all four zones of the olfactory epithelium are represented is shown in H–M . The expression pattern of robo-2 was compared with the expression pattern of specific olfactory epithelium zonal markers that include NQO-1 (zone I) ( I ), OCAM (zones II–IV) ( J ), M49 (zone II) ( K ), L45 (zone III) ( L ), and M50 (zone IV) ( M ). robo-2 is expressed in a high-to-low gradient in olfactory sensory neurons located in zones I (arrows) to IV (arrowheads), respectively. Regions of the olfactory epithelium expressing the different zonal markers are traced with a colored line on the apical surface of the olfactory epithelium to represent the four zones (zone I, magenta; zone II, green; zone III, yellow; zone IV, blue). S, Septum. Scale bars: A–E , H–M , 250 μm; F , G , 500 μm. N , Diagram representing the spatial relationship between the location of olfactory sensory neurons within the OE and their target glomeruli within the OB. Olfactory sensory neurons located in the dorsomedial regions of the olfactory epithelium (magenta) project axons to glomeruli in the dorsal region of the olfactory bulb, whereas olfactory sensory neurons in the ventrolateral region of the olfactory epithelium (blue and not shown) project axons to the ventral aspect of the olfactory bulb. D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal.

    Article Snippet: The sections were then blocked for 2 h in TNT (50 m m Tris-HCl, pH 7.6, 500 m m NaCl, and 0.5% Triton X-100) containing 10% fetal bovine serum (FBS) and incubated overnight with primary antibody at 4°C in TNT/10% FBS using the following dilutions: anti-OCAM at 1:100 (BD Biosciences, San Jose, CA), anti-NQO-1 at 1:100 (Abcam, Cambridge, MA), anti-SV2 (synaptic vesicle protein 2) at 1:1000 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Robo-2 at 1:350.

    Techniques: Expressing, In Situ Hybridization, Isolation, Mouse Assay

    Olfactory sensory neuron projections are disorganized in robo-2 −/− mice. Parasagittal ( A–F ) and coronal ( G–O ) sections of olfactory bulbs from P0 robo-2 +/+ ( A–C , G–I ) and robo-2 −/− ( D–F , J–O ) mice were stained with anti-NQO-1 ( A , C , D , F , G , I , J , L , M , O ) and anti-OCAM ( B , C , E , F , H , I , K , L , N , O ). In robo-2 −/− mice, NQO-1-expressing axons are restricted to the rostral and dorsal region of the olfactory bulbs ( A , G ), whereas OCAM-expressing axons target the ventral region of the olfactory bulbs ( B , H ). A subset of NQO-1-expressing axons is mistargeted to the ventral region of the olfactory bulbs in robo-2 −/− mice (arrowheads) ( D , F , J , L , M , O ). In addition, some regions of the ventral olfactory bulb that are innervated by OCAM-expressing axons in robo-2 +/+ mice lack innervation in robo-2 −/− mice (arrows). n = 9 robo-2 +/+ , 3 robo-2 +/− , and 11 robo-2 −/− . D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal. Scale bars, 250 μm.

    Journal: The Journal of Neuroscience

    Article Title: Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

    doi: 10.1523/JNEUROSCI.2217-07.2007

    Figure Lengend Snippet: Olfactory sensory neuron projections are disorganized in robo-2 −/− mice. Parasagittal ( A–F ) and coronal ( G–O ) sections of olfactory bulbs from P0 robo-2 +/+ ( A–C , G–I ) and robo-2 −/− ( D–F , J–O ) mice were stained with anti-NQO-1 ( A , C , D , F , G , I , J , L , M , O ) and anti-OCAM ( B , C , E , F , H , I , K , L , N , O ). In robo-2 −/− mice, NQO-1-expressing axons are restricted to the rostral and dorsal region of the olfactory bulbs ( A , G ), whereas OCAM-expressing axons target the ventral region of the olfactory bulbs ( B , H ). A subset of NQO-1-expressing axons is mistargeted to the ventral region of the olfactory bulbs in robo-2 −/− mice (arrowheads) ( D , F , J , L , M , O ). In addition, some regions of the ventral olfactory bulb that are innervated by OCAM-expressing axons in robo-2 +/+ mice lack innervation in robo-2 −/− mice (arrows). n = 9 robo-2 +/+ , 3 robo-2 +/− , and 11 robo-2 −/− . D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal. Scale bars, 250 μm.

    Article Snippet: The sections were then blocked for 2 h in TNT (50 m m Tris-HCl, pH 7.6, 500 m m NaCl, and 0.5% Triton X-100) containing 10% fetal bovine serum (FBS) and incubated overnight with primary antibody at 4°C in TNT/10% FBS using the following dilutions: anti-OCAM at 1:100 (BD Biosciences, San Jose, CA), anti-NQO-1 at 1:100 (Abcam, Cambridge, MA), anti-SV2 (synaptic vesicle protein 2) at 1:1000 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Robo-2 at 1:350.

    Techniques: Mouse Assay, Staining, Expressing

    Expression of NQO-1 and OCAM are unaltered in robo-2 −/− and slit-1 −/− ; slit-3 −/− mice. A–N , In situ hybridization of coronal sections of olfactory epithelia from P0 wild-type ( A , D , G , I , L ), slit-1 −/− ; slit-3 −/− ( B , E , H , J , M ), and robo-2 −/− ( C , F , K , N ) mice with cRNA probes specific for NQO-1 ( A–C ), OCAM ( D–F ), robo-2 ( G , H ), robo-1 ( I–K ), and rig-1 ( L–N ). robo-2 is expressed in a gradient in the OE with high levels of expression in the dorsomedial region of the OE and low levels in the ventrolateral region in wild-type ( G ) and slit-1 −/− ; slit-3 −/− ( H ) mice. NQO-1 is expressed in the dorsomedial region, whereas OCAM is expressed in the ventrolateral region of the olfactory epithelium in wild-type ( A , D ), slit-1 −/− ; slit-3 −/− ( B , E ), and robo-2 −/− ( C , F ) mice. Both robo-1 and rig-1 are not expressed in the olfactory epithelium in wild-type ( I , L ), slit-1 −/− ; slit-3 −/− ( J , M ), and robo-2 −/− ( K , N ) mice. n = 5 robo-2 −/− and n = 5 slit-1 −/− ; slit-3 −/− . O , P , In situ hybridization of parasagittal sections of olfactory bulbs from E16 slit-1 −/− embryos with cRNA probes specific for slit-3 ( O ) and slit-2 ( P ). Whereas slit-3 is expressed in the ventral region of the olfactory bulb, slit-2 is not expressed in the olfactory bulb of slit-1 −/− embryos. n = 3 slit-1 −/− . D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal. Scale bars, 250 μm.

    Journal: The Journal of Neuroscience

    Article Title: Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

    doi: 10.1523/JNEUROSCI.2217-07.2007

    Figure Lengend Snippet: Expression of NQO-1 and OCAM are unaltered in robo-2 −/− and slit-1 −/− ; slit-3 −/− mice. A–N , In situ hybridization of coronal sections of olfactory epithelia from P0 wild-type ( A , D , G , I , L ), slit-1 −/− ; slit-3 −/− ( B , E , H , J , M ), and robo-2 −/− ( C , F , K , N ) mice with cRNA probes specific for NQO-1 ( A–C ), OCAM ( D–F ), robo-2 ( G , H ), robo-1 ( I–K ), and rig-1 ( L–N ). robo-2 is expressed in a gradient in the OE with high levels of expression in the dorsomedial region of the OE and low levels in the ventrolateral region in wild-type ( G ) and slit-1 −/− ; slit-3 −/− ( H ) mice. NQO-1 is expressed in the dorsomedial region, whereas OCAM is expressed in the ventrolateral region of the olfactory epithelium in wild-type ( A , D ), slit-1 −/− ; slit-3 −/− ( B , E ), and robo-2 −/− ( C , F ) mice. Both robo-1 and rig-1 are not expressed in the olfactory epithelium in wild-type ( I , L ), slit-1 −/− ; slit-3 −/− ( J , M ), and robo-2 −/− ( K , N ) mice. n = 5 robo-2 −/− and n = 5 slit-1 −/− ; slit-3 −/− . O , P , In situ hybridization of parasagittal sections of olfactory bulbs from E16 slit-1 −/− embryos with cRNA probes specific for slit-3 ( O ) and slit-2 ( P ). Whereas slit-3 is expressed in the ventral region of the olfactory bulb, slit-2 is not expressed in the olfactory bulb of slit-1 −/− embryos. n = 3 slit-1 −/− . D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal. Scale bars, 250 μm.

    Article Snippet: The sections were then blocked for 2 h in TNT (50 m m Tris-HCl, pH 7.6, 500 m m NaCl, and 0.5% Triton X-100) containing 10% fetal bovine serum (FBS) and incubated overnight with primary antibody at 4°C in TNT/10% FBS using the following dilutions: anti-OCAM at 1:100 (BD Biosciences, San Jose, CA), anti-NQO-1 at 1:100 (Abcam, Cambridge, MA), anti-SV2 (synaptic vesicle protein 2) at 1:1000 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Robo-2 at 1:350.

    Techniques: Expressing, Mouse Assay, In Situ Hybridization

    Targeting of Robo-2-expressing olfactory sensory neuron axons in the olfactory bulb. A–L , Parasagittal sections of olfactory bulbs from E16 ( A–F ) and E18 ( G–L ) embryos stained with anti-Robo-2 ( A , C , D , F , G , I , J , L ), anti-NQO-1 ( B , C , H , I ), and anti-OCAM ( E , F , K , L ). At E16 and E18, olfactory sensory neuron axons expressing high levels of Robo-2 are observed in the dorsal and rostral regions of the OB ( A , D , G , J ). Robo-2 is also expressed at lower levels on a subset of axons targeting the ventral region of the olfactory bulb. NQO-1-expressing axons, originating from zone I of the olfactory epithelium, that target to the dorsal region of the OB express high levels of Robo-2 ( B , C , H , I ) (arrowheads). A subset of OCAM-expressing axons that originate from zones II–IV of the olfactory epithelium and target to the ventral region of the olfactory bulb do not express Robo-2 (arrow) ( E , F , K , L ). M–R , Coronal sections of olfactory bulbs from E18 embryos stained with anti-Robo-2 ( M , O , P , R ), anti-NQO-1 ( N , O ), and anti-OCAM ( Q , R ). At E18, olfactory sensory neuron axons expressing high levels of Robo-2 are observed in the dorsomedial regions of the OB (arrowheads), whereas lower levels of Robo-2 expression are observed on olfactory sensory neuron axons targeting to the ventrolateral region of the OB. As observed in sagittal sections ( G–L ), NQO-1-positive axons express high levels of Robo-2 (arrowheads) ( M–O ), whereas a subset of OCAM-positive axons do not express Robo-2 (arrows) ( P–R ). Robo-2-positive axons restricted to the nerve layer are marked with asterisks in P and R . D, Dorsal; V, ventral; R, rostral; C, caudal; M, medial; L, lateral. Scale bars, 250 μm.

    Journal: The Journal of Neuroscience

    Article Title: Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

    doi: 10.1523/JNEUROSCI.2217-07.2007

    Figure Lengend Snippet: Targeting of Robo-2-expressing olfactory sensory neuron axons in the olfactory bulb. A–L , Parasagittal sections of olfactory bulbs from E16 ( A–F ) and E18 ( G–L ) embryos stained with anti-Robo-2 ( A , C , D , F , G , I , J , L ), anti-NQO-1 ( B , C , H , I ), and anti-OCAM ( E , F , K , L ). At E16 and E18, olfactory sensory neuron axons expressing high levels of Robo-2 are observed in the dorsal and rostral regions of the OB ( A , D , G , J ). Robo-2 is also expressed at lower levels on a subset of axons targeting the ventral region of the olfactory bulb. NQO-1-expressing axons, originating from zone I of the olfactory epithelium, that target to the dorsal region of the OB express high levels of Robo-2 ( B , C , H , I ) (arrowheads). A subset of OCAM-expressing axons that originate from zones II–IV of the olfactory epithelium and target to the ventral region of the olfactory bulb do not express Robo-2 (arrow) ( E , F , K , L ). M–R , Coronal sections of olfactory bulbs from E18 embryos stained with anti-Robo-2 ( M , O , P , R ), anti-NQO-1 ( N , O ), and anti-OCAM ( Q , R ). At E18, olfactory sensory neuron axons expressing high levels of Robo-2 are observed in the dorsomedial regions of the OB (arrowheads), whereas lower levels of Robo-2 expression are observed on olfactory sensory neuron axons targeting to the ventrolateral region of the OB. As observed in sagittal sections ( G–L ), NQO-1-positive axons express high levels of Robo-2 (arrowheads) ( M–O ), whereas a subset of OCAM-positive axons do not express Robo-2 (arrows) ( P–R ). Robo-2-positive axons restricted to the nerve layer are marked with asterisks in P and R . D, Dorsal; V, ventral; R, rostral; C, caudal; M, medial; L, lateral. Scale bars, 250 μm.

    Article Snippet: The sections were then blocked for 2 h in TNT (50 m m Tris-HCl, pH 7.6, 500 m m NaCl, and 0.5% Triton X-100) containing 10% fetal bovine serum (FBS) and incubated overnight with primary antibody at 4°C in TNT/10% FBS using the following dilutions: anti-OCAM at 1:100 (BD Biosciences, San Jose, CA), anti-NQO-1 at 1:100 (Abcam, Cambridge, MA), anti-SV2 (synaptic vesicle protein 2) at 1:1000 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Robo-2 at 1:350.

    Techniques: Expressing, Staining

    Mistargeted axons of NQO-1-expressing olfactory sensory neurons innervate glomeruli in the ventral region of the olfactory bulb in slit and robo-2 mutant mice. Coronal sections at similar rostrocaudal levels of olfactory bulbs isolated from 8- to 12-week-old wild-type ( A , E , I ), slit-1 −/− ( B , F , J , M–O ), slit-1 −/− ; slit-3 −/− ( C , G , K ), and robo-2 c/c ; syn-1 Cre /+ ( D , H , L ) mice stained with anti-NQO-1 ( A–D , I–L , M–O ), anti-OCAM ( E–H , I–L , M , N ), anti-SV2 ( O ), and Hoechst ( A–O ). In wild-type mice, NQO-1-expressing axons innervate glomeruli restricted to the dorsomedial region of the olfactory bulb ( A , I ), and OCAM-expressing axons innervate glomeruli in the ventrolateral region of the olfactory bulb ( E , I ). In slit-1 −/− ( B , J ), slit-1 −/− ; slit-3 −/− ( C , K ), and robo-2 c/c ;syn-1 Cre /+ ( D , L ) mice, a subset of NQO-1-expressing axons are mistargeted to glomeruli in the ventral region of the olfactory bulb (arrows). NQO-1-expressing axons that target ectopically in the ventral region of the OB in slit-1 −/− mice form both homogenous (arrowheads) and heterogenous (arrows) glomeruli ( M , N ) and are positive for the presynaptic marker SV2, suggesting that they form synapses (arrowheads) ( O ). n = 10 wild type, n = 14 slit-1 −/− , n = 9 slit-1 −/− ; slit-3 −/− , and n = 4 robo-2 c/c ;syn-1 Cre /+ . Scale bars: A–L , 250 μm; M–O , 140 μm. P–R , Scatter plots showing the mapping of the positions of NQO-1-positive glomeruli in the olfactory bulb of an adult wild-type ( P ), slit-1 −/− ( Q ), and robo-2 c/c ;syn-1 Cre /+ ( R ) mouse. The relative positions of glomeruli containing NQO-1-positive axons were assessed in olfactory bulb sections isolated over a rostrocaudal distance of 1000 μm starting at 800 μm from the tip of the olfactory bulb. Although NQO-1-positive glomeruli are absent in the ventral region of the olfactory bulb (180° angle) from a wild-type mouse, NQO-1-positive glomeruli are consistently observed in the most ventral region of the OB in slit-1 −/− and robo-2 c/c ;syn-1 Cre /+ mice. Shown are representative plots from a single olfactory bulb for each genotype ( n = 4 olfactory bulbs from 2 mice of each genotype). D, Dorsal; V, ventral; L, lateral; M, medial.

    Journal: The Journal of Neuroscience

    Article Title: Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

    doi: 10.1523/JNEUROSCI.2217-07.2007

    Figure Lengend Snippet: Mistargeted axons of NQO-1-expressing olfactory sensory neurons innervate glomeruli in the ventral region of the olfactory bulb in slit and robo-2 mutant mice. Coronal sections at similar rostrocaudal levels of olfactory bulbs isolated from 8- to 12-week-old wild-type ( A , E , I ), slit-1 −/− ( B , F , J , M–O ), slit-1 −/− ; slit-3 −/− ( C , G , K ), and robo-2 c/c ; syn-1 Cre /+ ( D , H , L ) mice stained with anti-NQO-1 ( A–D , I–L , M–O ), anti-OCAM ( E–H , I–L , M , N ), anti-SV2 ( O ), and Hoechst ( A–O ). In wild-type mice, NQO-1-expressing axons innervate glomeruli restricted to the dorsomedial region of the olfactory bulb ( A , I ), and OCAM-expressing axons innervate glomeruli in the ventrolateral region of the olfactory bulb ( E , I ). In slit-1 −/− ( B , J ), slit-1 −/− ; slit-3 −/− ( C , K ), and robo-2 c/c ;syn-1 Cre /+ ( D , L ) mice, a subset of NQO-1-expressing axons are mistargeted to glomeruli in the ventral region of the olfactory bulb (arrows). NQO-1-expressing axons that target ectopically in the ventral region of the OB in slit-1 −/− mice form both homogenous (arrowheads) and heterogenous (arrows) glomeruli ( M , N ) and are positive for the presynaptic marker SV2, suggesting that they form synapses (arrowheads) ( O ). n = 10 wild type, n = 14 slit-1 −/− , n = 9 slit-1 −/− ; slit-3 −/− , and n = 4 robo-2 c/c ;syn-1 Cre /+ . Scale bars: A–L , 250 μm; M–O , 140 μm. P–R , Scatter plots showing the mapping of the positions of NQO-1-positive glomeruli in the olfactory bulb of an adult wild-type ( P ), slit-1 −/− ( Q ), and robo-2 c/c ;syn-1 Cre /+ ( R ) mouse. The relative positions of glomeruli containing NQO-1-positive axons were assessed in olfactory bulb sections isolated over a rostrocaudal distance of 1000 μm starting at 800 μm from the tip of the olfactory bulb. Although NQO-1-positive glomeruli are absent in the ventral region of the olfactory bulb (180° angle) from a wild-type mouse, NQO-1-positive glomeruli are consistently observed in the most ventral region of the OB in slit-1 −/− and robo-2 c/c ;syn-1 Cre /+ mice. Shown are representative plots from a single olfactory bulb for each genotype ( n = 4 olfactory bulbs from 2 mice of each genotype). D, Dorsal; V, ventral; L, lateral; M, medial.

    Article Snippet: The sections were then blocked for 2 h in TNT (50 m m Tris-HCl, pH 7.6, 500 m m NaCl, and 0.5% Triton X-100) containing 10% fetal bovine serum (FBS) and incubated overnight with primary antibody at 4°C in TNT/10% FBS using the following dilutions: anti-OCAM at 1:100 (BD Biosciences, San Jose, CA), anti-NQO-1 at 1:100 (Abcam, Cambridge, MA), anti-SV2 (synaptic vesicle protein 2) at 1:1000 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Robo-2 at 1:350.

    Techniques: Expressing, Mutagenesis, Mouse Assay, Isolation, Staining, Marker

    Slit-1 and Robo-2 are required for the segregation of zone I OSN axons to the dorsal region of the OB. Representation of zone I OSN projections in wild-type, slit-1 −/− , and robo-2 −/− mice. NQO-1-expressing zone I OSNs (magenta) located in the dorsal region of the OE project their axons to the dorsal aspect of the OB. The high dorsal to low ventral graded expression of Robo-2 (green) in OSN of the OE promotes the segregation of NQO-1-expressing axons to the dorsal region of the OB. Zone I OSN axons may be repelled by the Robo-2 ligand Slit-1, which is expressed in the ventral region of the OB. Loss of either Robo-2 or Slit-1 expression leads to mistargeting of a subset of zone I OSN axons to the ventral aspect of the OB.

    Journal: The Journal of Neuroscience

    Article Title: Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

    doi: 10.1523/JNEUROSCI.2217-07.2007

    Figure Lengend Snippet: Slit-1 and Robo-2 are required for the segregation of zone I OSN axons to the dorsal region of the OB. Representation of zone I OSN projections in wild-type, slit-1 −/− , and robo-2 −/− mice. NQO-1-expressing zone I OSNs (magenta) located in the dorsal region of the OE project their axons to the dorsal aspect of the OB. The high dorsal to low ventral graded expression of Robo-2 (green) in OSN of the OE promotes the segregation of NQO-1-expressing axons to the dorsal region of the OB. Zone I OSN axons may be repelled by the Robo-2 ligand Slit-1, which is expressed in the ventral region of the OB. Loss of either Robo-2 or Slit-1 expression leads to mistargeting of a subset of zone I OSN axons to the ventral aspect of the OB.

    Article Snippet: The sections were then blocked for 2 h in TNT (50 m m Tris-HCl, pH 7.6, 500 m m NaCl, and 0.5% Triton X-100) containing 10% fetal bovine serum (FBS) and incubated overnight with primary antibody at 4°C in TNT/10% FBS using the following dilutions: anti-OCAM at 1:100 (BD Biosciences, San Jose, CA), anti-NQO-1 at 1:100 (Abcam, Cambridge, MA), anti-SV2 (synaptic vesicle protein 2) at 1:1000 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Robo-2 at 1:350.

    Techniques: Mouse Assay, Expressing

    Loss of zonal targeting of NQO-1-expressing olfactory sensory neuron axons within the olfactory bulb in slit-1 −/− mice. A–M , Parasagittal sections of olfactory bulbs from P0 wild-type ( A–C ), slit-1 −/− ( D–F ), slit-3 −/− ( H–J ), and slit-1 −/− ; slit-3 −/− ( K–M ) mice stained with anti-NQO-1 ( A , C , D , F , H , J , K , M ) and anti-OCAM ( B , C , E , F , I , J , L , M ). In wild-type animals, NQO-1-expressing axons are restricted to the rostrodorsal region of the OB ( A , C ) and OCAM-expressing axons target to the ventral region of the olfactory bulb ( B , C ). Whereas NQO-1-expressing axons are properly targeted to the dorsal region of the olfactory bulb in slit-3 −/− mice ( H , J ), a subset of NQO-1-expressing axons mistarget to the most ventral region of the olfactory bulb in slit-1 −/− mice ( D , F ) (arrowheads). In slit-1 −/− ; slit-3 −/− mice, NQO-1-expressing axons are also observed in the ventral region of the olfactory bulb ( K , M ) (arrowheads). High-powered magnifications of ectopically projecting NQO-1-expressing axons (insets in D , F , K , M ) are shown in D′ , F′ , K′ , and M′ . n = 8 wild type, n = 5 slit-1 −/− , n = 8 slit-3 −/− , and n = 7 slit-1 −/− ; slit-3 −/− . N–P , Coronal sections at similar rostrocaudal levels of olfactory bulbs isolated from P0 ( N–P ) wild-type ( N ), slit-1 −/− ( O ), and slit-1 −/− ; slit-3 −/− ( P ) mice stained with anti-NQO-1 ( N–P ). In wild-type mice, NQO-1-expressing axons innervate the dorsomedial region of the olfactory bulb. In slit-1 −/− and slit-1 −/− ; slit-3 −/− mice, a subset of NQO-1-expressing axons are mistargeted to the ventral region of the olfactory bulb (arrows). n = 8 wild type, n = 8 slit-1 −/− , and n = 5 slit-1 −/− ; slit-3 −/− . D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal. Scale bars, 250 μm.

    Journal: The Journal of Neuroscience

    Article Title: Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

    doi: 10.1523/JNEUROSCI.2217-07.2007

    Figure Lengend Snippet: Loss of zonal targeting of NQO-1-expressing olfactory sensory neuron axons within the olfactory bulb in slit-1 −/− mice. A–M , Parasagittal sections of olfactory bulbs from P0 wild-type ( A–C ), slit-1 −/− ( D–F ), slit-3 −/− ( H–J ), and slit-1 −/− ; slit-3 −/− ( K–M ) mice stained with anti-NQO-1 ( A , C , D , F , H , J , K , M ) and anti-OCAM ( B , C , E , F , I , J , L , M ). In wild-type animals, NQO-1-expressing axons are restricted to the rostrodorsal region of the OB ( A , C ) and OCAM-expressing axons target to the ventral region of the olfactory bulb ( B , C ). Whereas NQO-1-expressing axons are properly targeted to the dorsal region of the olfactory bulb in slit-3 −/− mice ( H , J ), a subset of NQO-1-expressing axons mistarget to the most ventral region of the olfactory bulb in slit-1 −/− mice ( D , F ) (arrowheads). In slit-1 −/− ; slit-3 −/− mice, NQO-1-expressing axons are also observed in the ventral region of the olfactory bulb ( K , M ) (arrowheads). High-powered magnifications of ectopically projecting NQO-1-expressing axons (insets in D , F , K , M ) are shown in D′ , F′ , K′ , and M′ . n = 8 wild type, n = 5 slit-1 −/− , n = 8 slit-3 −/− , and n = 7 slit-1 −/− ; slit-3 −/− . N–P , Coronal sections at similar rostrocaudal levels of olfactory bulbs isolated from P0 ( N–P ) wild-type ( N ), slit-1 −/− ( O ), and slit-1 −/− ; slit-3 −/− ( P ) mice stained with anti-NQO-1 ( N–P ). In wild-type mice, NQO-1-expressing axons innervate the dorsomedial region of the olfactory bulb. In slit-1 −/− and slit-1 −/− ; slit-3 −/− mice, a subset of NQO-1-expressing axons are mistargeted to the ventral region of the olfactory bulb (arrows). n = 8 wild type, n = 8 slit-1 −/− , and n = 5 slit-1 −/− ; slit-3 −/− . D, Dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal. Scale bars, 250 μm.

    Article Snippet: The sections were then blocked for 2 h in TNT (50 m m Tris-HCl, pH 7.6, 500 m m NaCl, and 0.5% Triton X-100) containing 10% fetal bovine serum (FBS) and incubated overnight with primary antibody at 4°C in TNT/10% FBS using the following dilutions: anti-OCAM at 1:100 (BD Biosciences, San Jose, CA), anti-NQO-1 at 1:100 (Abcam, Cambridge, MA), anti-SV2 (synaptic vesicle protein 2) at 1:1000 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Robo-2 at 1:350.

    Techniques: Expressing, Mouse Assay, Staining, Isolation

    Deletion of renal tubular Keap1 from embryonic stage but not from adult stage leads to polyuria. ( a , b ) Control and Keap1-TKO mice were treated with DOX from adult stage. Urine volume ( a ) and relative transcript levels of Keap1 and Nqo1 genes in the kidney ( b ) of the mice are shown. ( c – f ) Control and Keap1-TKO mice were treated with DOX from embryonic stage. Urine volume ( c ), relative transcript levels of Keap1 and Nqo1 genes in the kidney ( d ), urine osmolality ( e ) and volume of drinking water consumed ( f ) by the mice are shown. Data of gene expression are the means±s.e. ( n =8). (* P

    Journal: Nature Communications

    Article Title: Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus

    doi: 10.1038/ncomms14577

    Figure Lengend Snippet: Deletion of renal tubular Keap1 from embryonic stage but not from adult stage leads to polyuria. ( a , b ) Control and Keap1-TKO mice were treated with DOX from adult stage. Urine volume ( a ) and relative transcript levels of Keap1 and Nqo1 genes in the kidney ( b ) of the mice are shown. ( c – f ) Control and Keap1-TKO mice were treated with DOX from embryonic stage. Urine volume ( c ), relative transcript levels of Keap1 and Nqo1 genes in the kidney ( d ), urine osmolality ( e ) and volume of drinking water consumed ( f ) by the mice are shown. Data of gene expression are the means±s.e. ( n =8). (* P

    Article Snippet: For immunostaining, samples were stained using anti-AQP2 (C-17, sc-9882; Santa Cruz; 1:500 dilution), AQP4 (Millipore; AB3594; 1:400 dilution), NCC (Millipore; AB3553; 1:1,000 dilution) and anti-NQO1 (ab2346, Abcam; 1:1,000 dilution), and positive reactivity was visualized with diaminobenzidine staining.

    Techniques: Mouse Assay, Expressing

    Generation of a mouse model that lacks Keap1 throughout the whole body and Nrf2 in the squamous epithelium. ( a ) Relative Nqo1 gene expression level compared with Hprt expression in the oesophagus, skin, liver, lung, kidney and heart of mice at 10 days of age. Data are the means±s.e. ( n =4). (* P

    Journal: Nature Communications

    Article Title: Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus

    doi: 10.1038/ncomms14577

    Figure Lengend Snippet: Generation of a mouse model that lacks Keap1 throughout the whole body and Nrf2 in the squamous epithelium. ( a ) Relative Nqo1 gene expression level compared with Hprt expression in the oesophagus, skin, liver, lung, kidney and heart of mice at 10 days of age. Data are the means±s.e. ( n =4). (* P

    Article Snippet: For immunostaining, samples were stained using anti-AQP2 (C-17, sc-9882; Santa Cruz; 1:500 dilution), AQP4 (Millipore; AB3594; 1:400 dilution), NCC (Millipore; AB3553; 1:1,000 dilution) and anti-NQO1 (ab2346, Abcam; 1:1,000 dilution), and positive reactivity was visualized with diaminobenzidine staining.

    Techniques: Expressing, Mouse Assay

    The β-lapachone (bL) compound suppresses the expression of BCSC markers in an NQO1-dependent manner. ( A – C ) The mRNA expression levels of DLGAP5, CD44, and ALDH1A1 were compared among MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) with or without bL (2 µM) over a 24-h treatment. GAPDH was used as an internal control, and each expression level was normalized to that of GAPDH . The data are presented as means ± SEM; n = 3; * p

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms19123813

    Figure Lengend Snippet: The β-lapachone (bL) compound suppresses the expression of BCSC markers in an NQO1-dependent manner. ( A – C ) The mRNA expression levels of DLGAP5, CD44, and ALDH1A1 were compared among MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) with or without bL (2 µM) over a 24-h treatment. GAPDH was used as an internal control, and each expression level was normalized to that of GAPDH . The data are presented as means ± SEM; n = 3; * p

    Article Snippet: Briefly, whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, and isolated total proteins were subjected to SDS-PAGE and membrane transfer for the incubation of the following primary antibodies (Ab): anti-NQO1 Ab (Abcam, Cambridge, United Kingdom), CD44 (Abcam, Cambridge, United Kingdom), ALDH1A1 (Abcam, Cambridge, United Kingdom), DLGAP5 (Abcam, Cambridge, United Kingdom), β-actin (Sigma-Aldrich, St. Louis, MO, USA), SIRT1 (Santa Cruz, Dallas, TX, USA), Lamin b (Santa Cruz, Dallas, TX, USA), and β-tubulin (AbFrontier, Seoul, South Korea).

    Techniques: Expressing, Multiple Displacement Amplification, Clone Assay

    Sirtuin 1 (SIRT1) is not involved in bL-NQO1-mediated cell death. ( A ) Cytoplasmic and nuclear fractionation was performed as described in Section 4 , 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.

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms19123813

    Figure Lengend Snippet: Sirtuin 1 (SIRT1) is not involved in bL-NQO1-mediated cell death. ( A ) Cytoplasmic and nuclear fractionation was performed as described in Section 4 , 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.

    Article Snippet: Briefly, whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, and isolated total proteins were subjected to SDS-PAGE and membrane transfer for the incubation of the following primary antibodies (Ab): anti-NQO1 Ab (Abcam, Cambridge, United Kingdom), CD44 (Abcam, Cambridge, United Kingdom), ALDH1A1 (Abcam, Cambridge, United Kingdom), DLGAP5 (Abcam, Cambridge, United Kingdom), β-actin (Sigma-Aldrich, St. Louis, MO, USA), SIRT1 (Santa Cruz, Dallas, TX, USA), Lamin b (Santa Cruz, Dallas, TX, USA), and β-tubulin (AbFrontier, Seoul, South Korea).

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

    The bL compound suppresses cell proliferation and migration in an NQO1-dependent manner. ( A ) Cell proliferation was determined by counting the cell numbers of MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) after treatment with bL (2 µM) for 24 h. The data are presented as means ± SEM; n = 3; ** p

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms19123813

    Figure Lengend Snippet: The bL compound suppresses cell proliferation and migration in an NQO1-dependent manner. ( A ) Cell proliferation was determined by counting the cell numbers of MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) after treatment with bL (2 µM) for 24 h. The data are presented as means ± SEM; n = 3; ** p

    Article Snippet: Briefly, whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, and isolated total proteins were subjected to SDS-PAGE and membrane transfer for the incubation of the following primary antibodies (Ab): anti-NQO1 Ab (Abcam, Cambridge, United Kingdom), CD44 (Abcam, Cambridge, United Kingdom), ALDH1A1 (Abcam, Cambridge, United Kingdom), DLGAP5 (Abcam, Cambridge, United Kingdom), β-actin (Sigma-Aldrich, St. Louis, MO, USA), SIRT1 (Santa Cruz, Dallas, TX, USA), Lamin b (Santa Cruz, Dallas, TX, USA), and β-tubulin (AbFrontier, Seoul, South Korea).

    Techniques: Migration, Multiple Displacement Amplification, Clone Assay

    The bL compound decreases endogenous ALDH1 activity. ( A ) The overall activity of ALDH1 was examined using an ALDEFLUOR assay in MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) treated with bL (2 µM) for 24 h. Green fluorescence was captured under a new hybrid microscope (Echo, San Diego, CA, USA). The scale bar is 130 μm. ( B ) For quantification, three independent areas were randomly taken, and cells with a lower intensity of ALDEFLUOR than the dimethyl sulfoxide (DMSO) control were counted. Quantitative analysis revealed that bL efficiently decreased the endogenous activity of ALDH1 in an NQO1-dependent manner. The yellow arrows indicate ALDEFLUOR (low) cells; * p

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms19123813

    Figure Lengend Snippet: The bL compound decreases endogenous ALDH1 activity. ( A ) The overall activity of ALDH1 was examined using an ALDEFLUOR assay in MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) treated with bL (2 µM) for 24 h. Green fluorescence was captured under a new hybrid microscope (Echo, San Diego, CA, USA). The scale bar is 130 μm. ( B ) For quantification, three independent areas were randomly taken, and cells with a lower intensity of ALDEFLUOR than the dimethyl sulfoxide (DMSO) control were counted. Quantitative analysis revealed that bL efficiently decreased the endogenous activity of ALDH1 in an NQO1-dependent manner. The yellow arrows indicate ALDEFLUOR (low) cells; * p

    Article Snippet: Briefly, whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, and isolated total proteins were subjected to SDS-PAGE and membrane transfer for the incubation of the following primary antibodies (Ab): anti-NQO1 Ab (Abcam, Cambridge, United Kingdom), CD44 (Abcam, Cambridge, United Kingdom), ALDH1A1 (Abcam, Cambridge, United Kingdom), DLGAP5 (Abcam, Cambridge, United Kingdom), β-actin (Sigma-Aldrich, St. Louis, MO, USA), SIRT1 (Santa Cruz, Dallas, TX, USA), Lamin b (Santa Cruz, Dallas, TX, USA), and β-tubulin (AbFrontier, Seoul, South Korea).

    Techniques: Activity Assay, Multiple Displacement Amplification, Clone Assay, Fluorescence, Microscopy

    The bL compound inhibits mammosphere formation. ( A ) MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) were induced to mammosphere formation for one and two weeks, as described in Section 4 . After mammosphere formation, bL (2 µM) was added for 24 h. Images were taken using a new hybrid microscope (Echo, San Diego, CA, USA). The scale bar is 200 μm. ( B ) Cell lysates obtained from the mammosphere formation assay were subjected to Western blot analysis using the indicated antibodies; β-tubulin was used as a loading control.

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms19123813

    Figure Lengend Snippet: The bL compound inhibits mammosphere formation. ( A ) MDA-MB-231 and two independent clones of NQO1 stable cells (NQO1 #1 and #2) were induced to mammosphere formation for one and two weeks, as described in Section 4 . After mammosphere formation, bL (2 µM) was added for 24 h. Images were taken using a new hybrid microscope (Echo, San Diego, CA, USA). The scale bar is 200 μm. ( B ) Cell lysates obtained from the mammosphere formation assay were subjected to Western blot analysis using the indicated antibodies; β-tubulin was used as a loading control.

    Article Snippet: Briefly, whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, and isolated total proteins were subjected to SDS-PAGE and membrane transfer for the incubation of the following primary antibodies (Ab): anti-NQO1 Ab (Abcam, Cambridge, United Kingdom), CD44 (Abcam, Cambridge, United Kingdom), ALDH1A1 (Abcam, Cambridge, United Kingdom), DLGAP5 (Abcam, Cambridge, United Kingdom), β-actin (Sigma-Aldrich, St. Louis, MO, USA), SIRT1 (Santa Cruz, Dallas, TX, USA), Lamin b (Santa Cruz, Dallas, TX, USA), and β-tubulin (AbFrontier, Seoul, South Korea).

    Techniques: Multiple Displacement Amplification, Clone Assay, Microscopy, Tube Formation Assay, Western Blot

    A : effect of pretreatment with polyethylene glycol (PEG)-catalase (1,000 U/ml) and N -acetylcysteine (NAC, 25 mmol/l) on high glucose (HG, 30 mmol/l, for 24 h)-induced changes in mRNA expression of Nqo1 , Gclc , and Hmox1 in cultured primary human CAECs.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia

    doi: 10.1152/ajpheart.00402.2010

    Figure Lengend Snippet: A : effect of pretreatment with polyethylene glycol (PEG)-catalase (1,000 U/ml) and N -acetylcysteine (NAC, 25 mmol/l) on high glucose (HG, 30 mmol/l, for 24 h)-induced changes in mRNA expression of Nqo1 , Gclc , and Hmox1 in cultured primary human CAECs.

    Article Snippet: To analyze protein expression of the Nrf2 targets NQO1, glutathione peroxidase (GPX), and GCLC, Western blotting was performed as described previously ( ) with the following primary antibodies: rabbit anti-GCLC (Abcam, ab41463; 1 μg/ml in 5% milk), rabbit anti-NQO1 (Abcam; ab34173, 1:2,000 in 5% milk), rabbit anti-GPX (Abcam, ab22604; 1:6,000 in 5% milk).

    Techniques: Expressing, Cell Culture

    The OE of HBC-specific Pax6 -knockout mice. (A–F) Images of hematoxylin and eosin-stained coronal sections from the OE of 6-week-old Pax6 flox/flox mice ( control; A) and K5-Cre +/− ::Pax6 flox/flox mice ( Pax6 -cKO; B) . (C–F) NQO1 was

    Journal: Stem Cells and Development

    Article Title: Horizontal Basal Cell-Specific Deletion of Pax6 Impedes Recovery of the Olfactory Neuroepithelium Following Severe Injury

    doi: 10.1089/scd.2015.0011

    Figure Lengend Snippet: The OE of HBC-specific Pax6 -knockout mice. (A–F) Images of hematoxylin and eosin-stained coronal sections from the OE of 6-week-old Pax6 flox/flox mice ( control; A) and K5-Cre +/− ::Pax6 flox/flox mice ( Pax6 -cKO; B) . (C–F) NQO1 was

    Article Snippet: The following primary antibodies were used: rabbit anti-Pax6 (1:1,000 [ ]), rabbit anti-Keratin5 (1:500, PRB-160P; Covance), rabbit anti-Ki-67 (1:500, NCL-Ki67p; Novocastra), goat anti-Sox2 (1:500, AF2018; R & D systems), goat anti-Sox10 (1:200, AF2864; R & D systems), goat anti-NQO1 (1:400, ab2346; Abcam), goat anti-OMP (1:1,000, 019-22291; Wako), armenian hamster anti-ICAM-1 (CD54) (1:200; BD Biosciences), and mouse anti-p63 (1:200, sc8431; Santa Cruz Biotechnology).

    Techniques: Knock-Out, Mouse Assay, Staining

    HBC-specific Pax6 -knockout severely impairs the long-term regeneration of the OE. (A, B) Images of NQO1-immunostained coronal sections of the OE from Pax6 flox/flox mice ( control; A) and K5-Cre +/− ::Pax6 flox/flox mice ( Pax6 -cKO; B) at 42 days postinjury

    Journal: Stem Cells and Development

    Article Title: Horizontal Basal Cell-Specific Deletion of Pax6 Impedes Recovery of the Olfactory Neuroepithelium Following Severe Injury

    doi: 10.1089/scd.2015.0011

    Figure Lengend Snippet: HBC-specific Pax6 -knockout severely impairs the long-term regeneration of the OE. (A, B) Images of NQO1-immunostained coronal sections of the OE from Pax6 flox/flox mice ( control; A) and K5-Cre +/− ::Pax6 flox/flox mice ( Pax6 -cKO; B) at 42 days postinjury

    Article Snippet: The following primary antibodies were used: rabbit anti-Pax6 (1:1,000 [ ]), rabbit anti-Keratin5 (1:500, PRB-160P; Covance), rabbit anti-Ki-67 (1:500, NCL-Ki67p; Novocastra), goat anti-Sox2 (1:500, AF2018; R & D systems), goat anti-Sox10 (1:200, AF2864; R & D systems), goat anti-NQO1 (1:400, ab2346; Abcam), goat anti-OMP (1:1,000, 019-22291; Wako), armenian hamster anti-ICAM-1 (CD54) (1:200; BD Biosciences), and mouse anti-p63 (1:200, sc8431; Santa Cruz Biotechnology).

    Techniques: Knock-Out, Mouse Assay

    The Nrf2-mediated induction of cytoprotective gene expression was elevated in the skin tissues of fat-1 transgenic and DHA-treated mice. Hairless fat-1 transgenic and WT mice ( n = 5 per treatment group) were maintained on AIN-93 diet supplemented with 10% safflower oil (rich in ω-6 fatty acids) for 5 weeks ( A – C ). ( A ) RT-PCR analysis was conducted to measure the mRNA levels of ho-1, nqo1 and nrf2 . ( B ) Collected skin tissues were placed on ice, and fat was removed to get an epidermal layer. The epidermal lysates and nuclear extracts from different groups were subjected to electrophoresis on SDS-PAGE and immunoblotted to detect protein expression of HO-1, NQO1 and Nrf2. ( C ) Paraffin-embedded skin tissue blocks were analyzed by IHC and the levels of Nrf2 (brown spots) were compared between hairless fat-1 transgenic and WT mice. Magnifications, ×100. Scale bar is 100 µm. ( D ) After 23 weeks of feeding the 10% safflower oil diet, whole tissue extracts (30 μg protein) were analyzed for the protein levels of Nrf2-regulated antioxidant gene by immunoblotting. Data are expressed as means ± SE. * p

    Journal: Scientific Reports

    Article Title: Endogenous ω-3 Fatty Acid Production by fat-1 Transgene and Topically Applied Docosahexaenoic Acid Protect against UVB-induced Mouse Skin Carcinogenesis

    doi: 10.1038/s41598-017-11443-2

    Figure Lengend Snippet: The Nrf2-mediated induction of cytoprotective gene expression was elevated in the skin tissues of fat-1 transgenic and DHA-treated mice. Hairless fat-1 transgenic and WT mice ( n = 5 per treatment group) were maintained on AIN-93 diet supplemented with 10% safflower oil (rich in ω-6 fatty acids) for 5 weeks ( A – C ). ( A ) RT-PCR analysis was conducted to measure the mRNA levels of ho-1, nqo1 and nrf2 . ( B ) Collected skin tissues were placed on ice, and fat was removed to get an epidermal layer. The epidermal lysates and nuclear extracts from different groups were subjected to electrophoresis on SDS-PAGE and immunoblotted to detect protein expression of HO-1, NQO1 and Nrf2. ( C ) Paraffin-embedded skin tissue blocks were analyzed by IHC and the levels of Nrf2 (brown spots) were compared between hairless fat-1 transgenic and WT mice. Magnifications, ×100. Scale bar is 100 µm. ( D ) After 23 weeks of feeding the 10% safflower oil diet, whole tissue extracts (30 μg protein) were analyzed for the protein levels of Nrf2-regulated antioxidant gene by immunoblotting. Data are expressed as means ± SE. * p

    Article Snippet: The membranes were incubated for 12–24 h at 4 °C with dilutions of primary antibodies against P-STAT3 (Cell Signaling Technology, Beverly, MA), lamin B, ubiquitin (Invitrogen, Carlsbad, CA), COX-2 (Cayman Chemical Co., Ann Arbor, MI), actin (Sigma Aldrich, St. Louis, MO), proteins modified with 4-HNE (JaICA, Nikken SEIL Co. Ltd., Shizuoka, Japan), HO-1 (Stressgen Biotechnologies Co., San Diego, CA), NQO1 (Abcam Inc., Cambridge, MA) and Nrf2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, SDS Page, Immunohistochemistry

    Nrf2-activators dose-dependently increase antioxidant protein expression in OLN-93 cells. HO-1 ( A ), NQO-1 ( B ) and p62 ( C ) protein expression levels after 24 h treatment in the OLN-93 oligodendrocyte cell line with 5 µM or 10 µM SFN, 45 µM or 90 µM MMF, 30 µg/mL or 60 µg/mL Protandim or their respective DMSO or EtOH vehicle control. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. All statistics reflect one-way ANOVA tests with post hoc Bonferroni correction; * p

    Journal: Antioxidants

    Article Title: Protandim Protects Oligodendrocytes against an Oxidative Insult

    doi: 10.3390/antiox5030030

    Figure Lengend Snippet: Nrf2-activators dose-dependently increase antioxidant protein expression in OLN-93 cells. HO-1 ( A ), NQO-1 ( B ) and p62 ( C ) protein expression levels after 24 h treatment in the OLN-93 oligodendrocyte cell line with 5 µM or 10 µM SFN, 45 µM or 90 µM MMF, 30 µg/mL or 60 µg/mL Protandim or their respective DMSO or EtOH vehicle control. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. All statistics reflect one-way ANOVA tests with post hoc Bonferroni correction; * p

    Article Snippet: Antibody Characterization Primary antibodies used were rat anti-MBP (catalog no. MCA409S; RRID: AB_325004; Abd Serotec, Oxfordshire, UK), rabbit anti-Olig2 (catalog no. AB9610; RRID: AB_570666; Millipore, Billerica, MA, USA), rabbit anti-HO-1 (catalog no. ADI-OSA-150F; RRID: AB_1505620; Enzo Life Sciences, Farmingdale, NY, USA), mouse anti-NQO-1 (catalog no. ab28947; RRID: AB_881738; Abcam, Cambridge, UK), and mouse anti-p62/SQSTM1 (catalog no. ab56416; RRID: AB_945626), mouse anti-β-actin (catalog no. A5441; RRID: AB_476744; Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Expressing, Western Blot

    Nrf2-activators dose-dependently increase antioxidant protein expression in mature primary rat OLs. HO-1, NQO-1 and p62 protein expression levels after 24 h treatment in mature primary rat OLs, differentiated for 7 days, with 5 µM SFN, 90 µM MMF,30 µg/mL Protandim or their respective DMSO or EtOH vehicle control. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect one-way ANOVA test with post hoc Bonferroni correction; * p

    Journal: Antioxidants

    Article Title: Protandim Protects Oligodendrocytes against an Oxidative Insult

    doi: 10.3390/antiox5030030

    Figure Lengend Snippet: Nrf2-activators dose-dependently increase antioxidant protein expression in mature primary rat OLs. HO-1, NQO-1 and p62 protein expression levels after 24 h treatment in mature primary rat OLs, differentiated for 7 days, with 5 µM SFN, 90 µM MMF,30 µg/mL Protandim or their respective DMSO or EtOH vehicle control. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect one-way ANOVA test with post hoc Bonferroni correction; * p

    Article Snippet: Antibody Characterization Primary antibodies used were rat anti-MBP (catalog no. MCA409S; RRID: AB_325004; Abd Serotec, Oxfordshire, UK), rabbit anti-Olig2 (catalog no. AB9610; RRID: AB_570666; Millipore, Billerica, MA, USA), rabbit anti-HO-1 (catalog no. ADI-OSA-150F; RRID: AB_1505620; Enzo Life Sciences, Farmingdale, NY, USA), mouse anti-NQO-1 (catalog no. ab28947; RRID: AB_881738; Abcam, Cambridge, UK), and mouse anti-p62/SQSTM1 (catalog no. ab56416; RRID: AB_945626), mouse anti-β-actin (catalog no. A5441; RRID: AB_476744; Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Expressing, Western Blot

    Autophagy induction of autophagy-related genes by cigarette smoke extract in vitro paralleled by upregulation of OSGIN1. Primary human airway basal cells were treated with cigarette smoke extract (CSE) in vitro. Increasing concentrations of CSE (0 to 10%) were assessed (n = 3/group). (A) TaqMan PCR assessment of gene expression changes induced by CSE. Oxidative stress genes HMOX1, CYP1A1 and NQO1 were used as positive control. Shown are autophagy-related genes, MAP1LC3B, SQSTM1 and GABARAPL1 , paralleled by OSGIN1 expression. Values are mean ± standard deviation; *, P

    Journal: Autophagy

    Article Title: Role of OSGIN1 in mediating smoking-induced autophagy in the human airway epithelium

    doi: 10.1080/15548627.2017.1301327

    Figure Lengend Snippet: Autophagy induction of autophagy-related genes by cigarette smoke extract in vitro paralleled by upregulation of OSGIN1. Primary human airway basal cells were treated with cigarette smoke extract (CSE) in vitro. Increasing concentrations of CSE (0 to 10%) were assessed (n = 3/group). (A) TaqMan PCR assessment of gene expression changes induced by CSE. Oxidative stress genes HMOX1, CYP1A1 and NQO1 were used as positive control. Shown are autophagy-related genes, MAP1LC3B, SQSTM1 and GABARAPL1 , paralleled by OSGIN1 expression. Values are mean ± standard deviation; *, P

    Article Snippet: The following primary antibodies were used: rabbit anti-human MAP1LC3B (1:200 for immunofluorescence, 1:800 for immunohistochemistry; Cell Signaling Technology, 3868); mouse anti-human LAMP1 (1:100; R & D Systems, MAB4800); mouse Anti-SQSTM1 (1:2000; BD Biosciences, 610833); mouse anti-human OSGIN1 (1:100; Sigma-Aldrich, SAB1407392), mouse anti-CD63 (1:100; Santa Cruz Biotechnology, sc-5275), mouse anti-NQO1 (1:200; Abcam, ab28947), mouse anti-HMOX1 (1:100; Thermo Scientific, MA1–112).

    Techniques: In Vitro, Polymerase Chain Reaction, Expressing, Positive Control, Standard Deviation

    Nrf2 promotes chemoresistance by inducing expression of ABCG2. (a and b) Kaplan-Meier curves of OS was plotted as a function of Nrf2 protein levels in GBC (a) and CC (b) patients, who had or had not received adjuvant chemotherapy. Nrf2 high, IHC score > 3; Nrf2 low, IHC score ≤ 3. The P value was calculated by a log-rank test. (c and d) Apoptosis analysis of GBC-SD (c) and QBC-939 (d) Cells in response to cisplatin when NFE2L2 was stably knockdown, or overexpressed, or when the cells were treated with Nrf2 agonists (t-BHQ 50 μM). n = 4; Bar, SEM. (e and f) Immunoblot and qPCR analysis of NQO1 and ABCG2 from paired GBC-SD (e up: sh-Con and sh- NFE2L2 ; e down: vector and NFE2L2 ) and QBC-939 (f up: sh-Con and sh- NFE2L2 ; f down: vector and NFE2L2 ). n = 3; Bar, SEM. (g and h) Immunofluorescence analysis of ABCG2 (green) and Nrf2 (red) from paired GBC-SD (g) and QBC-939 (h). DAPI (blue) serve as markers for nuclei. Representative images are shown. Original magnification, × 400; scale bars: 5 μm. * P

    Journal: EBioMedicine

    Article Title: Variants in oxidative stress-related genes affect the chemosensitivity through Nrf2-mediated signaling pathway in biliary tract cancer

    doi: 10.1016/j.ebiom.2019.08.037

    Figure Lengend Snippet: Nrf2 promotes chemoresistance by inducing expression of ABCG2. (a and b) Kaplan-Meier curves of OS was plotted as a function of Nrf2 protein levels in GBC (a) and CC (b) patients, who had or had not received adjuvant chemotherapy. Nrf2 high, IHC score > 3; Nrf2 low, IHC score ≤ 3. The P value was calculated by a log-rank test. (c and d) Apoptosis analysis of GBC-SD (c) and QBC-939 (d) Cells in response to cisplatin when NFE2L2 was stably knockdown, or overexpressed, or when the cells were treated with Nrf2 agonists (t-BHQ 50 μM). n = 4; Bar, SEM. (e and f) Immunoblot and qPCR analysis of NQO1 and ABCG2 from paired GBC-SD (e up: sh-Con and sh- NFE2L2 ; e down: vector and NFE2L2 ) and QBC-939 (f up: sh-Con and sh- NFE2L2 ; f down: vector and NFE2L2 ). n = 3; Bar, SEM. (g and h) Immunofluorescence analysis of ABCG2 (green) and Nrf2 (red) from paired GBC-SD (g) and QBC-939 (h). DAPI (blue) serve as markers for nuclei. Representative images are shown. Original magnification, × 400; scale bars: 5 μm. * P

    Article Snippet: The membranes were blocked with Tris-bufferred saline with 0.05% Tween 20 and 5% skimmed milk, and then the following antibodies were applied overnight at 4 °C: anti-GPX4, anti-GSR, and anti-Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CAT and anti-NQO1 (Abcam, Cambridge, MA, USA), anti-ABCG2 and anti-β-actin (Sigma, St. Louis, MO, USA).

    Techniques: Expressing, Immunohistochemistry, Stable Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Immunofluorescence

    GPX4, CAT, and GSR regulate Nrf2 protein expression through ROS. (a and b) Representative FACS profile of ROS levels, which was measured by H2DCFDA staining, in GBC-SD (a) and QBC-939 (b) when GPX4, CAT, or GSR was overexpressed or knockdown. (c and d) Analysis of correlation between the protein level of Nrf2 and the mRNA level of GPX4 (c, left) or GSR (c, right) was performed in 36 GBC tissues, and the mRNA level of CAT (d, left) or GSR (d, right) was performed in 52 CC tissues. Spearman's correlation was used. (e and f) Immunoblot and qPCR analysis of Nrf2, and it target NQO1 from GBC-SD transfected with scrambled siRNA (si-Con) or siRNA (si- GPX4 and si- GSR ) and empty vector, GPX4 , or GSR (e), and from QBC-939 transfected with si-Con or siRNA (si- CAT and si- GSR ) and empty vector or CAT or GSR (F). n = 3; Bar, SEM. (g and i) qPCR analysis of NFE2L2, NQO1 and ABCG2 expression in GBC-SD (g: si-Con, si- GPX4 and si- GSR ), and in QBC-939 (i: si-Con, si- CAT and si- GSR ) cells cultured in standard media supplemented with or without 10 mM NAC. n = 3; Bar, SEM. (h and j) Immunoblots of Nrf2, NQO1 and ABCG2 protein in GBC-SD (h: si-Con, si- GPX4 and si- GSR ), and in QBC-939 (j: si-Con, si- CAT and si- GSR ) cells incubated with or without 10 mM NAC. * P

    Journal: EBioMedicine

    Article Title: Variants in oxidative stress-related genes affect the chemosensitivity through Nrf2-mediated signaling pathway in biliary tract cancer

    doi: 10.1016/j.ebiom.2019.08.037

    Figure Lengend Snippet: GPX4, CAT, and GSR regulate Nrf2 protein expression through ROS. (a and b) Representative FACS profile of ROS levels, which was measured by H2DCFDA staining, in GBC-SD (a) and QBC-939 (b) when GPX4, CAT, or GSR was overexpressed or knockdown. (c and d) Analysis of correlation between the protein level of Nrf2 and the mRNA level of GPX4 (c, left) or GSR (c, right) was performed in 36 GBC tissues, and the mRNA level of CAT (d, left) or GSR (d, right) was performed in 52 CC tissues. Spearman's correlation was used. (e and f) Immunoblot and qPCR analysis of Nrf2, and it target NQO1 from GBC-SD transfected with scrambled siRNA (si-Con) or siRNA (si- GPX4 and si- GSR ) and empty vector, GPX4 , or GSR (e), and from QBC-939 transfected with si-Con or siRNA (si- CAT and si- GSR ) and empty vector or CAT or GSR (F). n = 3; Bar, SEM. (g and i) qPCR analysis of NFE2L2, NQO1 and ABCG2 expression in GBC-SD (g: si-Con, si- GPX4 and si- GSR ), and in QBC-939 (i: si-Con, si- CAT and si- GSR ) cells cultured in standard media supplemented with or without 10 mM NAC. n = 3; Bar, SEM. (h and j) Immunoblots of Nrf2, NQO1 and ABCG2 protein in GBC-SD (h: si-Con, si- GPX4 and si- GSR ), and in QBC-939 (j: si-Con, si- CAT and si- GSR ) cells incubated with or without 10 mM NAC. * P

    Article Snippet: The membranes were blocked with Tris-bufferred saline with 0.05% Tween 20 and 5% skimmed milk, and then the following antibodies were applied overnight at 4 °C: anti-GPX4, anti-GSR, and anti-Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CAT and anti-NQO1 (Abcam, Cambridge, MA, USA), anti-ABCG2 and anti-β-actin (Sigma, St. Louis, MO, USA).

    Techniques: Expressing, FACS, Staining, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Incubation

    GPX4, CAT, and GSR regulate chemosensitivity through Nrf2-mediated ABCG2 expression. (a and b) qPCR analysis of ABCG2 and NQO1 mRNA expression in paired GBC-SD (a) and QBC-939 (b) cells transfected with or without siRNA of GPX4, GSR, CAT . n = 3; Bar, SEM. (c and d) Immunoblots of ABCG2 and NQO1 in paired GBC-SD (c) and QBC-939 (d) cells that expressed GPX4, GSR , or CAT targeting siRNAs or a scrambled siRNA. (e and f) ABCG2-promoter luciferase assay in paired GBC-SD (a) and QBC-939 (b) cells that were transfected with or without siRNA of GPX4, GSR, CAT . n = 3; Bar, SEM. (g) The canonical sequence of the Nrf2-binding site (top, red), a potential Nrf2-binding site at -431 bp to -420 bp in the proximal promoter region of the human ABCG2 gene (middle, red), and introduced point mutations (bottom, green) used to inactivate the potential ABCG2-binding site are shown. (h and i) Determination of luciferase activity using vector only, wild type or mutant ABCG2 promoter in different pairs of GBC-SD (h) and QBC-939 (i) cells. (j) ChIP analysis of paired GBC-SD (sh-Con and sh- NFE2L2 ) cells immunoprecipitated by anti-Nrf2 or IgG antibody followed by qPCR using 2 primer sets for the Nrf2-binding site in the ABCG2 promoter or ABCG2 exon 1, respectively. Data represent the percent of input. n = 3; Bar, SEM. (k) Doxorubicin efflux of paired GBC-SD (left) and QBC-939 (right) cells were detected by FACS. * P

    Journal: EBioMedicine

    Article Title: Variants in oxidative stress-related genes affect the chemosensitivity through Nrf2-mediated signaling pathway in biliary tract cancer

    doi: 10.1016/j.ebiom.2019.08.037

    Figure Lengend Snippet: GPX4, CAT, and GSR regulate chemosensitivity through Nrf2-mediated ABCG2 expression. (a and b) qPCR analysis of ABCG2 and NQO1 mRNA expression in paired GBC-SD (a) and QBC-939 (b) cells transfected with or without siRNA of GPX4, GSR, CAT . n = 3; Bar, SEM. (c and d) Immunoblots of ABCG2 and NQO1 in paired GBC-SD (c) and QBC-939 (d) cells that expressed GPX4, GSR , or CAT targeting siRNAs or a scrambled siRNA. (e and f) ABCG2-promoter luciferase assay in paired GBC-SD (a) and QBC-939 (b) cells that were transfected with or without siRNA of GPX4, GSR, CAT . n = 3; Bar, SEM. (g) The canonical sequence of the Nrf2-binding site (top, red), a potential Nrf2-binding site at -431 bp to -420 bp in the proximal promoter region of the human ABCG2 gene (middle, red), and introduced point mutations (bottom, green) used to inactivate the potential ABCG2-binding site are shown. (h and i) Determination of luciferase activity using vector only, wild type or mutant ABCG2 promoter in different pairs of GBC-SD (h) and QBC-939 (i) cells. (j) ChIP analysis of paired GBC-SD (sh-Con and sh- NFE2L2 ) cells immunoprecipitated by anti-Nrf2 or IgG antibody followed by qPCR using 2 primer sets for the Nrf2-binding site in the ABCG2 promoter or ABCG2 exon 1, respectively. Data represent the percent of input. n = 3; Bar, SEM. (k) Doxorubicin efflux of paired GBC-SD (left) and QBC-939 (right) cells were detected by FACS. * P

    Article Snippet: The membranes were blocked with Tris-bufferred saline with 0.05% Tween 20 and 5% skimmed milk, and then the following antibodies were applied overnight at 4 °C: anti-GPX4, anti-GSR, and anti-Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CAT and anti-NQO1 (Abcam, Cambridge, MA, USA), anti-ABCG2 and anti-β-actin (Sigma, St. Louis, MO, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Luciferase, Sequencing, Binding Assay, Activity Assay, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation, FACS

    Dendrite extension of mitral cells (MCs) in the ΔD mutant. a Plasmid constructs for D-zone ablation (ΔD) are schematically illustrated. The DTA and Cre genes code for diphtheria toxin A and Cre recombinase, respectively. The NSE and OMACS promoters were used for region-specific expression of Cre and DTA in the olfactory epithelium (OE). The DTA gene is activated in the dorsal OE by the D-zone specific OMACS promoter, ablating D-zone OSNs. In the ΔD mutant, no NCAM signals were detected in the dorsal OE, and glomerular structures were absent in the dorsal olfactory bulb (OB). Sections were counterstained with DAPI. n = 3. D, dorsal; M, medial; V, ventral; L, lateral. Scale bars, 250 μm. b Dendrite maturation of MCs. The ratios (%) of MCs with branched dendrites are plotted for the WT and ΔD mice in the dorsal (D) and ventral (V) regions at postnatal day (P) 0, 7, and 14. c Detection of glomeruli in OB sections. OSN axons (green) and M/T cells (magenta) were immunostained with antibodies against OMP and Pcdh21, respectively. Note that glomerular structures are absent in the D-region OB of ΔD mice. Glomerular structures are encircled by dotted lines. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer. Scale bar, 100 μm. d Three-dimensional stack images of mitral-cell dendrites in the WT pThy1-YFP and ΔD pThy1-YFP mice at P21. Three OB regions, dorsal (D), ventral (V), and D-V border, were analyzed. To determine the D-V border (broken lines), sections were stained with anti-NQO1 (D region marker) antibodies. Note that near the border in the ΔD, some D-side MCs (indicated by magenta arrow heads) extend long primary dendrites crossing the border to the V-side glomeruli. Schematic drawings of ΔD OB sections are shown in the lower panel. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer. Scale bar, 100 μm. e A schematic diagram of dendrite extension of D-side MCs to the V-side glomeruli in the ΔD mice

    Journal: Communications Biology

    Article Title: Primary dendrites of mitral cells synapse unto neighboring glomeruli independent of their odorant receptor identity

    doi: 10.1038/s42003-018-0252-y

    Figure Lengend Snippet: Dendrite extension of mitral cells (MCs) in the ΔD mutant. a Plasmid constructs for D-zone ablation (ΔD) are schematically illustrated. The DTA and Cre genes code for diphtheria toxin A and Cre recombinase, respectively. The NSE and OMACS promoters were used for region-specific expression of Cre and DTA in the olfactory epithelium (OE). The DTA gene is activated in the dorsal OE by the D-zone specific OMACS promoter, ablating D-zone OSNs. In the ΔD mutant, no NCAM signals were detected in the dorsal OE, and glomerular structures were absent in the dorsal olfactory bulb (OB). Sections were counterstained with DAPI. n = 3. D, dorsal; M, medial; V, ventral; L, lateral. Scale bars, 250 μm. b Dendrite maturation of MCs. The ratios (%) of MCs with branched dendrites are plotted for the WT and ΔD mice in the dorsal (D) and ventral (V) regions at postnatal day (P) 0, 7, and 14. c Detection of glomeruli in OB sections. OSN axons (green) and M/T cells (magenta) were immunostained with antibodies against OMP and Pcdh21, respectively. Note that glomerular structures are absent in the D-region OB of ΔD mice. Glomerular structures are encircled by dotted lines. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer. Scale bar, 100 μm. d Three-dimensional stack images of mitral-cell dendrites in the WT pThy1-YFP and ΔD pThy1-YFP mice at P21. Three OB regions, dorsal (D), ventral (V), and D-V border, were analyzed. To determine the D-V border (broken lines), sections were stained with anti-NQO1 (D region marker) antibodies. Note that near the border in the ΔD, some D-side MCs (indicated by magenta arrow heads) extend long primary dendrites crossing the border to the V-side glomeruli. Schematic drawings of ΔD OB sections are shown in the lower panel. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer. Scale bar, 100 μm. e A schematic diagram of dendrite extension of D-side MCs to the V-side glomeruli in the ΔD mice

    Article Snippet: Antibodies used in this study are as follows: rabbit anti-CNG-A2 antibodies (1:200, Alomone Labs); rabbit anti-PSD95 antibodies (1:1000, Cell Signaling); mouse anti-Synaptophysin (Syn) antibodies (1:1000, Millipore); goat anti-OMP antibodies (1:1000, Wako Pure Chemical); rabbit anti-NQO1 antibodies (1:300, Abcam); guinea pig anti-vGlut2 antibodies (1:300, Millipore); rabbit anti-GluR1 antibodies (1:300, Abcam); rabbit anti-GFP antibodies (1:300, Invitrogen); chicken anti-GFP antibodies (1:300, Abcam).

    Techniques: Mutagenesis, Plasmid Preparation, Construct, Expressing, Mouse Assay, Staining, Marker

    β-Lap causes cell death and increases cellular radiosensitivity in NQO1 dependent manner. (A) Effects of β-lap on the apoptosis in wild type A549 cells and shQO1 A549 cells. (B) Effects of β-lap on the clonogenic survival of wild type A549 cells and shNQO1 A549 cells. An average of seven experiments ± SEM is shown.

    Journal: Experimental & Molecular Medicine

    Article Title: ?-Lapachone suppresses radiation-induced activation of nuclear factor-?B

    doi: 10.3858/emm.2010.42.5.034

    Figure Lengend Snippet: β-Lap causes cell death and increases cellular radiosensitivity in NQO1 dependent manner. (A) Effects of β-lap on the apoptosis in wild type A549 cells and shQO1 A549 cells. (B) Effects of β-lap on the clonogenic survival of wild type A549 cells and shNQO1 A549 cells. An average of seven experiments ± SEM is shown.

    Article Snippet: The anti-NF-κB p65 antibody was purchased from Cell Signaling Technology while anti-β-actin antibody was obtained from Sigma-Aldrich and the anti-NQO1 antibody was purchased from Zymed. β-Lap was purchased from Biomol.

    Techniques:

    Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P

    Journal: Molecular Medicine Reports

    Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

    doi: 10.3892/mmr.2018.9288

    Figure Lengend Snippet: Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P

    Article Snippet: To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

    Techniques: Expressing, Immunofluorescence, Staining, Western Blot

    Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P

    Journal: Molecular Medicine Reports

    Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

    doi: 10.3892/mmr.2018.9288

    Figure Lengend Snippet: Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P

    Article Snippet: To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

    Techniques: Expressing

    The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for NQO1 and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p

    Journal: PLoS ONE

    Article Title: NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

    doi: 10.1371/journal.pone.0009631

    Figure Lengend Snippet: The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for NQO1 and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p

    Article Snippet: Analysis of Nrf2 Activation Western blot analysis of nuclear protein fractions was performed by Odyssey system (LI-COR Bioscience, Nebraska USA), using antibodies specific for Nrf2, TBP, beta-actin (Santa Cruz Technology), NQO1 (Cell Signaling Technology).

    Techniques: Mouse Assay, Injection, Staining, MANN-WHITNEY, Two Tailed Test, Activation Assay, Western Blot

    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

    BL153 upregulated renal PGC-1 α and NQO1 expression. PGC-1 α and NQO1 expression was detected by western blot assay. n = 5; * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Magnolia Extract (BL153) Ameliorates Kidney Damage in a High Fat Diet-Induced Obesity Mouse Model

    doi: 10.1155/2013/367040

    Figure Lengend Snippet: BL153 upregulated renal PGC-1 α and NQO1 expression. PGC-1 α and NQO1 expression was detected by western blot assay. n = 5; * P

    Article Snippet: The latter were blocked with 5% milk, followed by incubation with the following antibodies: TNF-α , PGC-1α (Abcam, Cambridge, MA), PAI-1 (BD Bioscience, San Jose, CA), 3-NT (Millipore, Billerica, MA), 4-HNE (Alpha Diagnostic International, San Antonio, TX), HK II, β -actin, and NQO1 (SantaCruz Biotechnology, Santa Cruz, CA).

    Techniques: Pyrolysis Gas Chromatography, Expressing, Western Blot

    Western blot analysis of NQO1, γ-GCS, and HO-1 levels in the lungs of bleomycin-induced pulmonary fibrosis rats. The decreased levels of NQO1, γ-GCS, and HO-1 protein expression were significantly increased by administration of BMSCs.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Bone marrow mesenchymal stem cells protect against bleomycin-induced pulmonary fibrosis in rat by activating Nrf2 signaling

    doi:

    Figure Lengend Snippet: Western blot analysis of NQO1, γ-GCS, and HO-1 levels in the lungs of bleomycin-induced pulmonary fibrosis rats. The decreased levels of NQO1, γ-GCS, and HO-1 protein expression were significantly increased by administration of BMSCs.

    Article Snippet: The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline with Tween (TTBS) for 1 h and probed with primary antibodies against NRF2 (1:400 dilution, Boster, Wuhan, China), KEAP1 (1:400 dilution, Boster, Wuhan, China), γ-GCS (1:400 dilution; Boster, Wuhan, China), HO-1 (1:400 dilution; Bioss Biotech, Beijing, China), and NQO1 (1:1000 dilution; Proteintech, Wuhan, China) overnight at 4°C.

    Techniques: Western Blot, Expressing

    NQO1 regulates HIF-1α stability in cancer cells. ( a , b ) NQO1 increases the stability of HIF-1α protein under normoxia. Cells were transfected with pHIF-1α-myc-His 6 and incubated with 10 μg ml −1 cycloheximide for 3 min, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( a ) and MDA-MB-231 ( b ) cells. β-actin was used as the internal control. ( c , d ) NQO1 prolongs the half-life of HIF-1α protein under hypoxia. Cells were transfected with pHIF-1α-myc-His 6 , exposed to 0.5% O 2 for 2 h, incubated with 10 μg ml −1 cycloheximide for 2 h, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( c ) and MDA-MB-231 ( d ) cells. β-actin was used as the internal control. HIF-1α protein levels were quantified using Image J, and band intensities were normalized to those of β-actin (band intensity at t 0 was defined as 100%).

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 regulates HIF-1α stability in cancer cells. ( a , b ) NQO1 increases the stability of HIF-1α protein under normoxia. Cells were transfected with pHIF-1α-myc-His 6 and incubated with 10 μg ml −1 cycloheximide for 3 min, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( a ) and MDA-MB-231 ( b ) cells. β-actin was used as the internal control. ( c , d ) NQO1 prolongs the half-life of HIF-1α protein under hypoxia. Cells were transfected with pHIF-1α-myc-His 6 , exposed to 0.5% O 2 for 2 h, incubated with 10 μg ml −1 cycloheximide for 2 h, and then harvested at the indicated times. The protein levels of HIF-1α and NQO1 were examined by immunoblot analysis in RKO ( c ) and MDA-MB-231 ( d ) cells. β-actin was used as the internal control. HIF-1α protein levels were quantified using Image J, and band intensities were normalized to those of β-actin (band intensity at t 0 was defined as 100%).

    Article Snippet: The slides were then incubated overnight at 4 °C with anti-NQO1 (NBP-1-31355, Novus) and anti-HIF-1α (sc-13515, Sata Cruz Biotechnology), as indicated.

    Techniques: Transfection, Incubation, Multiple Displacement Amplification

    NQO1 enhances HIF-1α expression. ( a , b ) RKO cells ( a ) and MDA-MB-231 cells ( b ) were exposed to 0.5% O 2 for 8 h and harvested at the indicated times. Whole-cell lysates were analysed by immunoblotting for HIF-1α, HIF-1β, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( c , d ) Various concentrations of pshNQO1 and pNQO1-myc-His 6 (0–10 μg) were transiently transfected into RKO ( c ) and MDA-MB-231 ( d ) cells, respectively. After 48 h, the cells were exposed to 20 or 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. EV indicates pshCont ( c ) and pCDNA3.1-myc-His 6 ( d ). SE, short exposure; LE, long exposure. ( e ) pNQO1-myc-His 6 was transiently transfected into RKO/pshNQO1 cells. After 48 h, the cells were exposed to 0.5% O 2 for 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( f ) Mouse embryonic fibroblast wild type and NQO1 −/− cells were exposed to 20 or 0.5% O 2 for 2 h and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure.

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 enhances HIF-1α expression. ( a , b ) RKO cells ( a ) and MDA-MB-231 cells ( b ) were exposed to 0.5% O 2 for 8 h and harvested at the indicated times. Whole-cell lysates were analysed by immunoblotting for HIF-1α, HIF-1β, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( c , d ) Various concentrations of pshNQO1 and pNQO1-myc-His 6 (0–10 μg) were transiently transfected into RKO ( c ) and MDA-MB-231 ( d ) cells, respectively. After 48 h, the cells were exposed to 20 or 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. EV indicates pshCont ( c ) and pCDNA3.1-myc-His 6 ( d ). SE, short exposure; LE, long exposure. ( e ) pNQO1-myc-His 6 was transiently transfected into RKO/pshNQO1 cells. After 48 h, the cells were exposed to 0.5% O 2 for 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure. ( f ) Mouse embryonic fibroblast wild type and NQO1 −/− cells were exposed to 20 or 0.5% O 2 for 2 h and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. SE, short exposure; LE, long exposure.

    Article Snippet: The slides were then incubated overnight at 4 °C with anti-NQO1 (NBP-1-31355, Novus) and anti-HIF-1α (sc-13515, Sata Cruz Biotechnology), as indicated.

    Techniques: Expressing, Multiple Displacement Amplification, Transfection

    NQO1 stabilizes HIF-1α through inhibiting ubiquitination and proteasome-mediated degradation. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively. The cells were then exposed to 0.5% O 2 for 2 h, and incubated for 1 h in the presence or absence of MG132. Whole-cell extracts were immunoprecipitated with an anti- HIF-1α antibody, and ubiquitinated HIF-1α was detected with an anti-ubiquitin antibody. ( c , d ) RKO ( c ) and MDA-MB-231 ( d ) cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were treated with or without cycloheximide and MG132, incubated for 1 h, and harvested. The collected cells were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. ( e , f ) RKO cells ( e ) and MDA-MB-231 cells ( f ) were exposed to 0.5% O 2 . After 2 h, the cells were harvested and immunoprecipitated with anti- HIF-1α or anti-IgG (negative control) antibodies. The precipitates were analysed by immunoblotting with the indicated antibodies. ( g ) RKO/pshCont and RKO/pshNQO1 cells were treated with or without 1 mM of DMOG for 1 h, and then exposed to 0.5% O 2 . After 2 h, the cells were harvested and analysed by immunoblotting with the indicated antibodies. ( h ) RKO/pshCont and RKO/pshNQO1 cells were transfected with siPHD1, siPHD2, siPHD3 or HIF-1α. After 48 h, the cells were treated with 50 μg MG132 for 6 h, exposed to 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, hydroxylated-HIF-1α, PHD1, PHD2, PHD3, NQO1 and β-actin. X-ray films were exposed for 1, 3 or 5 min to detect the signals of total HIF-1α, hydroxylated-HIF-1α.

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 stabilizes HIF-1α through inhibiting ubiquitination and proteasome-mediated degradation. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively. The cells were then exposed to 0.5% O 2 for 2 h, and incubated for 1 h in the presence or absence of MG132. Whole-cell extracts were immunoprecipitated with an anti- HIF-1α antibody, and ubiquitinated HIF-1α was detected with an anti-ubiquitin antibody. ( c , d ) RKO ( c ) and MDA-MB-231 ( d ) cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were treated with or without cycloheximide and MG132, incubated for 1 h, and harvested. The collected cells were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. ( e , f ) RKO cells ( e ) and MDA-MB-231 cells ( f ) were exposed to 0.5% O 2 . After 2 h, the cells were harvested and immunoprecipitated with anti- HIF-1α or anti-IgG (negative control) antibodies. The precipitates were analysed by immunoblotting with the indicated antibodies. ( g ) RKO/pshCont and RKO/pshNQO1 cells were treated with or without 1 mM of DMOG for 1 h, and then exposed to 0.5% O 2 . After 2 h, the cells were harvested and analysed by immunoblotting with the indicated antibodies. ( h ) RKO/pshCont and RKO/pshNQO1 cells were transfected with siPHD1, siPHD2, siPHD3 or HIF-1α. After 48 h, the cells were treated with 50 μg MG132 for 6 h, exposed to 0.5% O 2 for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, hydroxylated-HIF-1α, PHD1, PHD2, PHD3, NQO1 and β-actin. X-ray films were exposed for 1, 3 or 5 min to detect the signals of total HIF-1α, hydroxylated-HIF-1α.

    Article Snippet: The slides were then incubated overnight at 4 °C with anti-NQO1 (NBP-1-31355, Novus) and anti-HIF-1α (sc-13515, Sata Cruz Biotechnology), as indicated.

    Techniques: Multiple Displacement Amplification, Transfection, Incubation, Immunoprecipitation, Negative Control

    NQO1 interacts with HIF-1α in cytosol. ( a ) RKO cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were harvested and lysates were co-immunoprecipitated with anti-HIF-1α (left) or anti-NQO1 (right) antibody and anti-IgG antibody as a negative control. Then, the precipitates were analysed by immunoblot analysis with anti -HIF-1α and -NQO1 antibodies. ( b ) RKO cells were transfected with HIF-1α-myc-His 6 (left) or NQO1-myc-His 6 (right), subjected to Ni-NTA bead-based pulldown, and analysed by immunoblotting with anti-His 6 , -HIF-1α and -NQO1 antibodies. ( c ) Illustration of HIF-1α domain. ( d , e ) GFP pulldown assays were performed with purified EGFP fusion proteins containing the indicated amino-acid residues of HIF-1α, along with whole-cell lysates from RKO cells. The immunoprecipitated proteins were analysed by immunoblotting with anti-GFP and -NQO1 antibodies. ( f ) Extracted cytosolic or nuclear protein was immunoprecipitated with anti- HIF-1α (upper panel) or anti-NQO1 (lower panel) antibodies and analysed by immunoblotting to detect HIF-1α and NQO1.

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 interacts with HIF-1α in cytosol. ( a ) RKO cells were exposed to 20 or 0.5% O 2 . After 2 h, the cells were harvested and lysates were co-immunoprecipitated with anti-HIF-1α (left) or anti-NQO1 (right) antibody and anti-IgG antibody as a negative control. Then, the precipitates were analysed by immunoblot analysis with anti -HIF-1α and -NQO1 antibodies. ( b ) RKO cells were transfected with HIF-1α-myc-His 6 (left) or NQO1-myc-His 6 (right), subjected to Ni-NTA bead-based pulldown, and analysed by immunoblotting with anti-His 6 , -HIF-1α and -NQO1 antibodies. ( c ) Illustration of HIF-1α domain. ( d , e ) GFP pulldown assays were performed with purified EGFP fusion proteins containing the indicated amino-acid residues of HIF-1α, along with whole-cell lysates from RKO cells. The immunoprecipitated proteins were analysed by immunoblotting with anti-GFP and -NQO1 antibodies. ( f ) Extracted cytosolic or nuclear protein was immunoprecipitated with anti- HIF-1α (upper panel) or anti-NQO1 (lower panel) antibodies and analysed by immunoblotting to detect HIF-1α and NQO1.

    Article Snippet: The slides were then incubated overnight at 4 °C with anti-NQO1 (NBP-1-31355, Novus) and anti-HIF-1α (sc-13515, Sata Cruz Biotechnology), as indicated.

    Techniques: Immunoprecipitation, Negative Control, Transfection, Purification

    NQO1 increases transcriptional activity of HIF-1α in cancer cells under hypoxia. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively, exposed to 20 or 0.5% O 2 for 8 h, and then harvested. Quantitative PCR was used to amplify PDK1, PGK1 and LDHA. The signals were normalized by 18S rRNA . (mean±s.d. shown) n =3 in each group. * P

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 increases transcriptional activity of HIF-1α in cancer cells under hypoxia. ( a , b ) RKO ( a ) and MDA-MB-231 ( b ) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His 6 or pNQO1-myc-His 6 , respectively, exposed to 20 or 0.5% O 2 for 8 h, and then harvested. Quantitative PCR was used to amplify PDK1, PGK1 and LDHA. The signals were normalized by 18S rRNA . (mean±s.d. shown) n =3 in each group. * P

    Article Snippet: The slides were then incubated overnight at 4 °C with anti-NQO1 (NBP-1-31355, Novus) and anti-HIF-1α (sc-13515, Sata Cruz Biotechnology), as indicated.

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction

    Upregulation of NQO1 correlates with poor prognosis and expression of HIF-1α in colorectal cancer. ( a ) An Oncomine analysis of the TCGA colorectal database indicated that NQO1 expressions are elevated in colorectal cancers ( n =102) compared with normal colorectal tissues ( n =19). **** P

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: Upregulation of NQO1 correlates with poor prognosis and expression of HIF-1α in colorectal cancer. ( a ) An Oncomine analysis of the TCGA colorectal database indicated that NQO1 expressions are elevated in colorectal cancers ( n =102) compared with normal colorectal tissues ( n =19). **** P

    Article Snippet: The slides were then incubated overnight at 4 °C with anti-NQO1 (NBP-1-31355, Novus) and anti-HIF-1α (sc-13515, Sata Cruz Biotechnology), as indicated.

    Techniques: Expressing

    NQO1 promotes in vivo tumour growth. ( a ) RKO/pNQO1, RKO/shCont and RKO/pshNQO1 cells were injected subcutaneously into the right flank of athymic, 7-week-old female BALB/C nude mice, and tumour growth was assessed. Tumour volume (TV) was calculated by using the following formula: TV=length × (width) 2 × 0.5. Each group contained 12 animals. (** P

    Journal: Nature Communications

    Article Title: NQO1 inhibits proteasome-mediated degradation of HIF-1α

    doi: 10.1038/ncomms13593

    Figure Lengend Snippet: NQO1 promotes in vivo tumour growth. ( a ) RKO/pNQO1, RKO/shCont and RKO/pshNQO1 cells were injected subcutaneously into the right flank of athymic, 7-week-old female BALB/C nude mice, and tumour growth was assessed. Tumour volume (TV) was calculated by using the following formula: TV=length × (width) 2 × 0.5. Each group contained 12 animals. (** P

    Article Snippet: The slides were then incubated overnight at 4 °C with anti-NQO1 (NBP-1-31355, Novus) and anti-HIF-1α (sc-13515, Sata Cruz Biotechnology), as indicated.

    Techniques: In Vivo, Injection, Mouse Assay

    Accumulation of Nrf2 in the nuclear fraction in df-SH-SY5Y cells. (A) Cells were treated with or without 200 μM Na 2 S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na 2 S) for 2 h, and Nrf2 protein levels in nuclear fractions were analyzed by western blotting. Lamin B1 was used as nuclear loading control (bottom panel). (B) Cells were treated with or without 200 μM Na 2 S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na 2 S) for 10 h, and NQO1 protein levels in cytosol fractions were analyzed by western blotting. β-actin was used as cytosol loading control (bottom panel). (C) Cells were treated with 0, 100 and 200 μM Na 2 S or BS-Mix (prepared by 200 μM Na 2 S) for 2 h, and GSH levels were measured using HPLC-FL with ABD-F derivatized samples. Values indicate means±S.D. * P

    Journal: Redox Biology

    Article Title: Cysteine persulfides and polysulfides produced by exchange reactions with H2S protect SH-SY5Y cells from methylglyoxal-induced toxicity through Nrf2 activation

    doi: 10.1016/j.redox.2017.03.020

    Figure Lengend Snippet: Accumulation of Nrf2 in the nuclear fraction in df-SH-SY5Y cells. (A) Cells were treated with or without 200 μM Na 2 S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na 2 S) for 2 h, and Nrf2 protein levels in nuclear fractions were analyzed by western blotting. Lamin B1 was used as nuclear loading control (bottom panel). (B) Cells were treated with or without 200 μM Na 2 S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na 2 S) for 10 h, and NQO1 protein levels in cytosol fractions were analyzed by western blotting. β-actin was used as cytosol loading control (bottom panel). (C) Cells were treated with 0, 100 and 200 μM Na 2 S or BS-Mix (prepared by 200 μM Na 2 S) for 2 h, and GSH levels were measured using HPLC-FL with ABD-F derivatized samples. Values indicate means±S.D. * P

    Article Snippet: Next, the membrane was incubated with primary antibodies Nrf2 (H-300; Santa Cruz Biotechnology, CA, USA), Lamin B1 (Cell Signaling Technology, Danvers, MA, USA) or NQO1 (Abnova Co., Taipei City, Taiwan), β-actin (Wako, Osaka, Japan) washed with PBS, and incubated with secondary antibodies HRP-conjugated goat anti-mouse IgG antibody or HRP-conjugated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA).

    Techniques: Western Blot, High Performance Liquid Chromatography

    Western blot analysis of Nrf2 target proteins. A, After 28 days of diet, Western blot of 3 random animals from each experimental group was performed for the prototypical Nrf2-dependent proteins: GCLM, GstA2, and Nqo1. B, Densitometry of the presented blot demonstrated significant increases in the examined Nrf2-dependent proteins in the AlbCre+/caNrf2+ animal when compared with AlbCre+/caNrf2− animals independent of diet. * P

    Journal: Toxicological Sciences

    Article Title: Activation of the Nrf2-ARE Pathway in Hepatocytes Protects Against Steatosis in Nutritionally Induced Non-alcoholic Steatohepatitis in Mice

    doi: 10.1093/toxsci/kfu184

    Figure Lengend Snippet: Western blot analysis of Nrf2 target proteins. A, After 28 days of diet, Western blot of 3 random animals from each experimental group was performed for the prototypical Nrf2-dependent proteins: GCLM, GstA2, and Nqo1. B, Densitometry of the presented blot demonstrated significant increases in the examined Nrf2-dependent proteins in the AlbCre+/caNrf2+ animal when compared with AlbCre+/caNrf2− animals independent of diet. * P

    Article Snippet: Gels were transferred to PVDF membranes and probed overnight with anti-NQO1 (Epitomics), anti-GCLM (provided by Dr Terry Kavanagh, University of Washington, Seattle, WA), anti-GstA2, anti-MTTP (Abcam), anti α-SMA (Abcam), or anti-β-actin (Abcam) antibodies.

    Techniques: Western Blot

    Nrf2 and downstream target proteins are decreased in YG8R DRGs. (A) Representative immunoblots of Nrf2, Hmox, Nqo1, and Sod2 in the YG8R DRGs. Each lane represents an individual mouse. (B) Densitometry of blots shown in A, normalized to tubulin. Each

    Journal: Antioxidants & Redox Signaling

    Article Title: Frataxin Deficiency Leads to Defects in Expression of Antioxidants and Nrf2 Expression in Dorsal Root Ganglia of the Friedreich's Ataxia YG8R Mouse Model

    doi: 10.1089/ars.2012.4537

    Figure Lengend Snippet: Nrf2 and downstream target proteins are decreased in YG8R DRGs. (A) Representative immunoblots of Nrf2, Hmox, Nqo1, and Sod2 in the YG8R DRGs. Each lane represents an individual mouse. (B) Densitometry of blots shown in A, normalized to tubulin. Each

    Article Snippet: After electrophoresis, the proteins were transferred to nitrocellulose membranes by the iBlot device (Invitrogen), blocked with an Odyssey blocking buffer (LI-COR Biotechnology) for 1 h, and incubated overnight with the following primary antibodies in a blocking buffer: frataxin, Nrf2 (C-20 for mouse and H-300 for human; Santa Cruz Biotech, Santa Cruz, CA); Nrf2 (Epitomics); Nrf2 for ND7/23 cells [a kind gift from Dr. McMahon ( )]; tubulin (Sigma); Gstm1, Mbp, TxnRD, and S100a8 (Abcam); Grlx1 (R & D Systems); Prx3 (AbFrontier); Nqo1 (Epitomics), Hmox and Sod2 (Stressgen/Enzo), and Lamin B1 (Proteintech).

    Techniques: Western Blot

    Linagliptin activated the NRF2 antioxidant pathway. The expression levels of the KEAP1, nuclear NRF2, HO-1 and NQO1 were determined by Western blot (A). The binding capacity of NRF2 to ARE was assessed by EMSA (B).The data were presented as mean ± SD (n=6 for each group. *P

    Journal: Experimental Animals

    Article Title: Linagliptin protects rat carotid artery from balloon injury and activates the NRF2 antioxidant pathway

    doi: 10.1538/expanim.18-0089

    Figure Lengend Snippet: Linagliptin activated the NRF2 antioxidant pathway. The expression levels of the KEAP1, nuclear NRF2, HO-1 and NQO1 were determined by Western blot (A). The binding capacity of NRF2 to ARE was assessed by EMSA (B).The data were presented as mean ± SD (n=6 for each group. *P

    Article Snippet: Subsequently, the membranes were blocked with 5% non-fat milk and incubated with primary antibodies against NRF2 (1:1,000; Abcam, Cambridge, MA, USA), KEAP1 (1:1,000; Abcam), HO-1 (1:1,000; Abcam), NQO1 (1:1,000; BOSTER, Wuhan, China), β-actin (1:500; Bioss, Beijing, China), Histone H3 (1:500; Bioss) at 4°C overnight.

    Techniques: Expressing, Western Blot, Binding Assay