anti nrf2 antibody Abcam Search Results


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  • 90
    Abcam antibodies against nrf2
    H 2 O 2 causes an increased association of <t>NRF2</t> mRNA with ribosomes. Shown are data for HEK293 cells after treatment with 100 μM H 2 O 2 (A) or various doses of H 2 O 2 (B) for 10 min and harvesting 1 h later. RNA was isolated from polysomes (A and B)
    Antibodies Against Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti nrf2
    Decitabine and GO-203 combination decreases expression of DNMT1, 3b, increases Nox4, Duox2, activates <t>Nrf2</t> and Smad signaling pathway leading to downregulation of c-Myc A–B, H9 and HuT-78 cells were left untreated, treated with 3 uM GO-203 each day for 3 days, single dose of 40 nM Decitabine or the combination. Cells were harvested at 96 hours. Lysates were immunoblotted with the indicated antibodies.
    Anti Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 729 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam antibodies against phospho nrf2
    Interleukin-6 activates the <t>Keap1/Nrf2</t> system in irradiated OSCC cells. ( A ) The images of immunofluorescence of Nrf2 in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. ( B ) The protein levels of Nrf2, phospho-Nrf2, and phospho-STAT3 in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. The whole-cell and nuclear protein were prepared, and the expression of Nrf2, phospho-Nrf2, and phospho-STAT3 proteins was examined using a western blot analysis. ( C ) The expression of Keap1 mRNA in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. ( D ) The protein levels of Keap1 and phospho-p62 in SAS cells. The western blot analysis was done using the same lysate as B . ( E ) The images of immunofluoresent double staining of Keap1 and phospho-p62 in SAS cells at 48 h after 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. The arrows indicate the activated phospho-p62. ( F ) The Nrf2/Keap1 complex formation detected by an immunoprecipitation analysis in SAS cells. After the same treatment as B , the cell lysates were immunoprecipitated using anti-Keap1 antibody. The immunoprecipitates were immunoblotted with anti-Nrf2 antibody.
    Antibodies Against Phospho Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology nrf2
    Morphological plasticity is increased in A549 cells stably expressing control or <t>Nrf2</t> shRNA. A microfabricated μ-Taur device, was used to assay a cell's ability to migrate through narrow 3-diamensional channels of various dimensions 16 hrs after loading. A) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 12 micron posts. Inset shows close ups of posts; B) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 8 micron posts. Inset shows close ups of posts; C D) Migration and plasticity of cells stably expressing Nrf2 shRNA in channels with 12 micron posts (C) or 8 micron posts (D).
    Nrf2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 5745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti padpr
    Morphological plasticity is increased in A549 cells stably expressing control or <t>Nrf2</t> shRNA. A microfabricated μ-Taur device, was used to assay a cell's ability to migrate through narrow 3-diamensional channels of various dimensions 16 hrs after loading. A) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 12 micron posts. Inset shows close ups of posts; B) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 8 micron posts. Inset shows close ups of posts; C D) Migration and plasticity of cells stably expressing Nrf2 shRNA in channels with 12 micron posts (C) or 8 micron posts (D).
    Anti Padpr, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam polyclonal anti nrf2
    Morphological plasticity is increased in A549 cells stably expressing control or <t>Nrf2</t> shRNA. A microfabricated μ-Taur device, was used to assay a cell's ability to migrate through narrow 3-diamensional channels of various dimensions 16 hrs after loading. A) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 12 micron posts. Inset shows close ups of posts; B) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 8 micron posts. Inset shows close ups of posts; C D) Migration and plasticity of cells stably expressing Nrf2 shRNA in channels with 12 micron posts (C) or 8 micron posts (D).
    Polyclonal Anti Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti nrf2
    Effects of SFN on the expression of <t>Nrf2,</t> HO-1, NQO1, CD11b/c, and MOR in the prefrontal cortex from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein expression in the prefrontal cortex from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P
    Rabbit Anti Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti nrf2
    Effects of SFN on the expression of <t>Nrf2,</t> HO-1, NQO1, CD11b/c, and MOR in the prefrontal cortex from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein expression in the prefrontal cortex from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P
    Rabbit Polyclonal Anti Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit p nrf2
    Effects of SFN on the expression of <t>Nrf2,</t> HO-1, NQO1, CD11b/c, and MOR in the prefrontal cortex from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein expression in the prefrontal cortex from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P
    Rabbit P Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti nrf2 primary antibody
    Paeonin exerted an anti-oxidative role by PI3K/Akt-mediated <t>Nrf2</t> signaling pathway. ( A ) Cell viability was tested by MTT assay in H 2 O 2 -incubated GES-1 cells with 200 μg/ml Paeonin for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. The cell viability presented a time-dependent increase. ( B ) PI3K/Akt-mediated Keap-1 Nrf2 signaling pathway-related protein expressions were measured by WB in H 2 O 2 -exposed GES-1 cells with 200 μg/ml Paeonin for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. ( C ) The location expression of Nrf2 was examined by IF in H 2 O 2 -stimulated GES-1 cells with 200 μg/ml Paeonin for 2 h, 8 h and 12 h. The cell nucleus was stained into blue, while Nrf2 protein was stained into red. ( D ) GSH content was detected in the GSE-1, H 2 O 2 , H 2 O 2 + Paeonin and H 2 O 2 + Paeonin + GSK690693 groups. GSH content in the three experiment groups was higher than that in the GSE-1 group; * P
    Rabbit Anti Nrf2 Primary Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit monoclonal anti nrf2
    Roles of FA and Bz in the exhibition of cell proliferation and the promotion of EMT or in the prevention of apoptosis. ① FA and Bz activate the intracellular ROS of JEG-3, ② which in turn activates oxidative stress. ③ This activates apoptosis by activating apoptosis-related eIF2, or by activating the antioxidant factor, <t>Nrf2,</t> ④ to regulate the activation of ROS by inducing an antioxidant effect through the increase of Nrf2 which blocks the activation of ROS. However, FA and Bz not only activate ROS, but also increase the activity of Nrf2, which prevents the activation of oxidative stress and apoptosis. ⑤ In addition, FA and Bz induce cell proliferation and EMT, either directly or indirectly through Nrf2. Therefore, FA and Bz inhibit apoptosis through the ROS-Nrf2 pathway, leading to an increase in proliferation and EMT.
    Rabbit Monoclonal Anti Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti nrf2 solution
    The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) exhibited an increased <t>Nrf2</t> expression when evaluated 6 months after exposure to GWI-related chemicals and stress. Figures (A1–A4) illustrate examples of neuron specific nuclear antigen positive (NeuN+) hippocampal CA3 pyramidal neurons (green) displaying nuclear translocation of Nrf2 (red particles) in a naïve control animal (arrows in A1,A2 ) and in an animal exposed to GWI-related chemicals and stress (arrows in B1,B2 ). Note that nuclear translocation of Nrf2 is increased in the animal exposed to GWI-related chemicals and stress (B1,B2) . Bar charts in C1–C3 compare percentages of NeuN+ hippocampal neurons displaying nuclear Nrf2 (C1) , percentages of hippocampal neurons showing robust nuclear Nrf2 (C2) and the extent of activated Nrf2 measured through ELISA in the entire hippocampus between age-matched naïve control animals and animals exposed to GWI-related chemicals and stress ( n = 5–6/group). Scale bar, (A1,B1) , 10 μm; (A2,B2) , 5 μm. ∗ p
    Rabbit Anti Nrf2 Solution, supplied by Abcam, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti human mouse nrf2
    Oxidative stress induces T reg cell apoptosis in the tumor environment. ( a ) The effect of ovarian cancer ascites on human T reg cell apoptosis. Mouse T reg cells and conventional T cells (T conv ) were cocultured with 50% human ovarian cancer ascites or hydrogen peroxide for 24 h. Additional cultures were treated with N -acetyl-cysteine (NAC) as a free radical scavenger. Annexin V + T reg cells and T conv cells were analyzed by flow cytometry. n = 5. ( b,c ) The effect of ovarian cancer ascites on proapoptotic and antiapoptotic gene transcripts in T reg cells. T reg cells were exposed to ascites or ascites plus NAC after 24 h. Proapoptotic ( b ) and antiapoptotic ( c ) transcripts were quantified by real-time PCR. n = 3. ( d,e ) The effect of hydrogen peroxide on proapoptotic and antiapoptotic transcripts in T reg cells. T reg cells were exposed to hydrogen peroxide or hydrogen peroxide plus NAC after 24 h. Proapoptotic ( d ) and antiapoptotic ( e ) mouse gene transcripts were quantified by real-time PCR. n = 3. ( f,g ) Expression of mouse <t>NRF2</t> and NRF2-associated gene transcripts ( f ) and proteins ( g ) in T cell subsets as determined by real-time PCR and immunoblotting, respectively. n = 5. ( h ) The expression of intracellular ROS in T reg cells. T reg cells and conventional T cells were cultured overnight. Amounts of intracellular ROS were measured by flow cytometry. n = 5. MFI, mean fluorescence intensity. ( i ) The effect of NRF2 inducers on T reg cell apoptosis in vitro . Mouse T reg cells were cultured for 24 h with 50% ascites or medium containing H 2 O 2 . NRF2 inducers diethylmaleate (DEM; 100 μM) and sulforaphane (10 μM) were added to the culture for 24 h. Controls were treated with vehicle (DMSO). Apoptosis was measured by flow cytometry with annexin V. n = 4. ( j – l ) The effect of sulforaphane on tumor immunity. MC38-bearing mice were treated with sulforaphane (25 mg/kg, 5 d per week) or vehicle (DMSO). Tumor T reg cell apoptosis ( j ) and CD8 + T cell polyfunctional cytokine expression ( k ) were analyzed by flow cytometry. Tumor volume was monitored ( l ). n = 10. All data are shown as the mean and s.d. and are from 1 experiment with 10 animals per group ( i – l ) or from single experiments with cells isolated from individual animal for each data point ( a – h ). * P
    Anti Human Mouse Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti rat nrf2 monoclonal antibody
    Keap-1 and <t>Nrf2</t> expression in different tissues measured via western blot analysis. The expression of Keap-1 and Nrf2 in the (A) heart, (B) lung and (C) kidney tissue. All data are presented as the mean ± standard deviation and were obtained from at least six independent experiments or tests. 1, Normal control group; 2, Normal obese group; 3, Normal asthma group; 4, Obese + asthma group; 5, RSV obese group; 6, RSV asthma group; 7, RSV obese + asthma group; RSV, resveratrol; Keap-1, kelch-like ECH associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2.
    Rabbit Anti Rat Nrf2 Monoclonal Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti nrf2
    Ferulic acid induces <t>Nrf2</t> activation and translocation into the nucleus. (A–D) Representative images from four independent immunofluorescence experiments in which we performed a double-labeling with DAPI (A 1 –D 1 ) and an anti-Nrf2 antibody (A 2 –D 2 ) . Merged images are shown in (A 3 –D 3 ) . XZ and YZ cross-sections in the boxes (referred to the dashed lines) from the confocal Z -stack acquisitions illustrate cytosolic or nuclear fluorescence signal(s) (XZ and YZ boxes: a 1 –d 1 refer to DAPI staining; a 2 –d 2 : refer to Nrf2 fluorescence; a 3 –d 3 : Merge). In cells treated with TMT, a strong Nrf2 activation was detected which, however, remains mainly confined in the cytoplasm (b 1 –b 3 in B) . In TMT + FA treated cells, there was a further cytoplasmic enhancement of Nrf2 expression compared to TMT alone which also translocates into the nucleus as indicated by Z -stack acquisitions (c 1 –c 3 in C) . FA administration (10 μM) induced an endogenous antioxidant response leading to a strong increase in Nrf2 expression in the cytosol (d 1 –d 3 in D) compared to Control (a 1 -a 3 in A) . Scale bar: 20 μm. For further information see text.
    Mouse Anti Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti rat nrf2 polyclonal
    Ferulic acid induces <t>Nrf2</t> activation and translocation into the nucleus. (A–D) Representative images from four independent immunofluorescence experiments in which we performed a double-labeling with DAPI (A 1 –D 1 ) and an anti-Nrf2 antibody (A 2 –D 2 ) . Merged images are shown in (A 3 –D 3 ) . XZ and YZ cross-sections in the boxes (referred to the dashed lines) from the confocal Z -stack acquisitions illustrate cytosolic or nuclear fluorescence signal(s) (XZ and YZ boxes: a 1 –d 1 refer to DAPI staining; a 2 –d 2 : refer to Nrf2 fluorescence; a 3 –d 3 : Merge). In cells treated with TMT, a strong Nrf2 activation was detected which, however, remains mainly confined in the cytoplasm (b 1 –b 3 in B) . In TMT + FA treated cells, there was a further cytoplasmic enhancement of Nrf2 expression compared to TMT alone which also translocates into the nucleus as indicated by Z -stack acquisitions (c 1 –c 3 in C) . FA administration (10 μM) induced an endogenous antioxidant response leading to a strong increase in Nrf2 expression in the cytosol (d 1 –d 3 in D) compared to Control (a 1 -a 3 in A) . Scale bar: 20 μm. For further information see text.
    Rabbit Anti Rat Nrf2 Polyclonal, supplied by Abcam, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti rat nrf2 antibody
    Effects of ferulic acid (FA) and dimethylbiguanide (DMBG) on Keap-1 and <t>Nrf2</t> expression in hepatocytes and cardiomyocytes. (a) Western blot results. (b) Keap-1 and Nrf2 expression levels in hepatocytes. (c) Keap-1 and Nrf2 expression levels in cardiomyocytes. Mean±SEM, n =3. ## p
    Rabbit Anti Rat Nrf2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti mouse nrf2 antibody
    SFN exerts its renoprotective role via the activation of <t>Nrf2</t> in HK2 cells. (a) Cells were treated for 48 h with control or Nrf2 siRNA (the transfection efficiency was measured by western blot analysis). (b) SFN did not increase the Nrf2 nuclear protein level in Nrf2-deficient cells. (c) SFN did not increase the HO-1 protein level in Nrf2-deficient cells. (d) SFN did not increase cell viability in Nrf2-deficient cells. (e) SFN did not decrease reactive oxygen species in Nrf2-deficient cells. ∗ P
    Rabbit Anti Mouse Nrf2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 83/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti nf e2 related factor 2 antibody
    SFN exerts its renoprotective role via the activation of <t>Nrf2</t> in HK2 cells. (a) Cells were treated for 48 h with control or Nrf2 siRNA (the transfection efficiency was measured by western blot analysis). (b) SFN did not increase the Nrf2 nuclear protein level in Nrf2-deficient cells. (c) SFN did not increase the HO-1 protein level in Nrf2-deficient cells. (d) SFN did not increase cell viability in Nrf2-deficient cells. (e) SFN did not decrease reactive oxygen species in Nrf2-deficient cells. ∗ P
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    Abcam anti nrf2 antibody ep1808y bsa and azide free
    SFN exerts its renoprotective role via the activation of <t>Nrf2</t> in HK2 cells. (a) Cells were treated for 48 h with control or Nrf2 siRNA (the transfection efficiency was measured by western blot analysis). (b) SFN did not increase the Nrf2 nuclear protein level in Nrf2-deficient cells. (c) SFN did not increase the HO-1 protein level in Nrf2-deficient cells. (d) SFN did not increase cell viability in Nrf2-deficient cells. (e) SFN did not decrease reactive oxygen species in Nrf2-deficient cells. ∗ P
    Anti Nrf2 Antibody Ep1808y Bsa And Azide Free, supplied by Abcam, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit monoclonal anti phospho nrf2
    Trehalose inhibited H 2 O 2 -induced increase of intracellular ROS. (A) Statistical analysis showed that trehalose pretreatment prevented H 2 O 2 -induced increase of intracellular ROS. (B) Western blotting demonstrated that trehalose did not inhibit the upregulated expression of <t>Nrf2</t> and phosphorylated Nrf2 caused by H 2 O 2 . (C and D)Enzyme activity assay revealed that trehalose suppressed H 2 O 2 -induced reduction in CAT and SOD. The values are expressed as mean±SEM (n=5 per group). *: p
    Rabbit Monoclonal Anti Phospho Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti nuclear factor erythroid 2 related factor 2
    Trehalose inhibited H 2 O 2 -induced increase of intracellular ROS. (A) Statistical analysis showed that trehalose pretreatment prevented H 2 O 2 -induced increase of intracellular ROS. (B) Western blotting demonstrated that trehalose did not inhibit the upregulated expression of <t>Nrf2</t> and phosphorylated Nrf2 caused by H 2 O 2 . (C and D)Enzyme activity assay revealed that trehalose suppressed H 2 O 2 -induced reduction in CAT and SOD. The values are expressed as mean±SEM (n=5 per group). *: p
    Anti Nuclear Factor Erythroid 2 Related Factor 2, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam ser40 antibody
    SFN up-regulates the expression and function of Nrf2 in the heart. Mice were treated as described in Fig. 1 . Nrf2 expression at mRNA and protein levels was detected by real-time PCR ( A ) and Western blot ( B ), respectively. The activation of Nrf2 was examined by immunohistochemical staining ( C , counter stain with hematoxylin, bar = 25μm) and Western blot ( D ) for Nrf2 phosphorylation at <t>Ser40</t> (p-Nrf2). Nrf2 function was measured by determining the expression of Nrf2 downstream genes, catalase (CAT), NAD(P)H: quinone oxidoreductase (NQO1), and heme oxygenase 1 (HO-1), at both mRNA ( E ) and protein ( F ) levels with real-time PCR and Western blot, respectively. Data are presented as the mean ± SD (n = 7). *, p
    Ser40 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Abcam nuclear factor erythroid 2 related factor nrf2
    SFN up-regulates the expression and function of Nrf2 in the heart. Mice were treated as described in Fig. 1 . Nrf2 expression at mRNA and protein levels was detected by real-time PCR ( A ) and Western blot ( B ), respectively. The activation of Nrf2 was examined by immunohistochemical staining ( C , counter stain with hematoxylin, bar = 25μm) and Western blot ( D ) for Nrf2 phosphorylation at <t>Ser40</t> (p-Nrf2). Nrf2 function was measured by determining the expression of Nrf2 downstream genes, catalase (CAT), NAD(P)H: quinone oxidoreductase (NQO1), and heme oxygenase 1 (HO-1), at both mRNA ( E ) and protein ( F ) levels with real-time PCR and Western blot, respectively. Data are presented as the mean ± SD (n = 7). *, p
    Nuclear Factor Erythroid 2 Related Factor Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Abcam monoclonal rabbit anti phospho ser40 nrf2
    SFN up-regulates the expression and function of Nrf2 in the heart. Mice were treated as described in Fig. 1 . Nrf2 expression at mRNA and protein levels was detected by real-time PCR ( A ) and Western blot ( B ), respectively. The activation of Nrf2 was examined by immunohistochemical staining ( C , counter stain with hematoxylin, bar = 25μm) and Western blot ( D ) for Nrf2 phosphorylation at <t>Ser40</t> (p-Nrf2). Nrf2 function was measured by determining the expression of Nrf2 downstream genes, catalase (CAT), NAD(P)H: quinone oxidoreductase (NQO1), and heme oxygenase 1 (HO-1), at both mRNA ( E ) and protein ( F ) levels with real-time PCR and Western blot, respectively. Data are presented as the mean ± SD (n = 7). *, p
    Monoclonal Rabbit Anti Phospho Ser40 Nrf2, supplied by Abcam, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam nrf2 primary antibodies
    Effects of genistein on GPR30 expression and reactive oxygen species (ROS) generation. (A) MDA-MB-231 and HCC1937 cells were treated with 50 μM Genistein. After 48 hours of incubation, the mRNA expression of GPR30 was assessed by reverse transcription-PCR. (B) The intracellular ROS levels in genistein treated MDA-MB-231 and HCC1937 cells were measured using a DCF-DA fluorescent dye. Cells were exposed to either dimethyl sulfoxide or 50 μM genistein for 24 hours. Images of cellular fluorescence were acquired by using a live cell image microscope. (C) The protein level of <t>Nrf2</t> in HCC1937 with and without genistein treatment for 72 hours was measured by Western blot analysis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase .
    Nrf2 Primary Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Abcam rabbit antihuman nrf2 antibody
    Phase II genes expression in A2780 cells after CA and DMCA treatment in the presence of catalase. (a) <t>Nrf2</t> mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); (b) GSTP1 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); (c) HO-1 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); n =3; data represented as mean±SD.
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    Image Search Results


    H 2 O 2 causes an increased association of NRF2 mRNA with ribosomes. Shown are data for HEK293 cells after treatment with 100 μM H 2 O 2 (A) or various doses of H 2 O 2 (B) for 10 min and harvesting 1 h later. RNA was isolated from polysomes (A and B)

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: H 2 O 2 causes an increased association of NRF2 mRNA with ribosomes. Shown are data for HEK293 cells after treatment with 100 μM H 2 O 2 (A) or various doses of H 2 O 2 (B) for 10 min and harvesting 1 h later. RNA was isolated from polysomes (A and B)

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Isolation

    NRF2 5′-UTR activation by H 2 O 2 treatment. The dicistronic pRL-Nrf2 5′UTR-FL reporter construct was transfected into HEK293 cells. Transfected cells were treated with various doses of H 2 O 2 (A) or 100 μM H 2 O 2 (B) before harvesting

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: NRF2 5′-UTR activation by H 2 O 2 treatment. The dicistronic pRL-Nrf2 5′UTR-FL reporter construct was transfected into HEK293 cells. Transfected cells were treated with various doses of H 2 O 2 (A) or 100 μM H 2 O 2 (B) before harvesting

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Activation Assay, Construct, Transfection

    G-quadruplex sequence-dependent NRF2 5′-UTR activation by H 2 O 2 in cells. A dicistronic luciferase reporter construct of the wild-type human NRF2 5′ UTR (555 nt) or a mutant NRF2 5′ UTR with GGGG at nt −195 to −192

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: G-quadruplex sequence-dependent NRF2 5′-UTR activation by H 2 O 2 in cells. A dicistronic luciferase reporter construct of the wild-type human NRF2 5′ UTR (555 nt) or a mutant NRF2 5′ UTR with GGGG at nt −195 to −192

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Sequencing, Activation Assay, Luciferase, Construct, Mutagenesis

    Methylation and footprinting to define G-quadruplex structure. (A) The 31-mer fragments corresponding to the region spanning nt −198 to −168 of the wild-type or mutant NRF2 5′ UTR were treated with DMS to determine the monomeric

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: Methylation and footprinting to define G-quadruplex structure. (A) The 31-mer fragments corresponding to the region spanning nt −198 to −168 of the wild-type or mutant NRF2 5′ UTR were treated with DMS to determine the monomeric

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Methylation, Footprinting, Mutagenesis

    H 2 O 2 dose-dependent increase of EF1a interaction with NRF2 mRNA in HEK293 cells. HEK293 cells were treated with various doses of H 2 O 2 for 10 min and harvested 1 h later. (A) Cytoplasmic extracts were incubated with biotinylated 31-mer wild-type or A4

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: H 2 O 2 dose-dependent increase of EF1a interaction with NRF2 mRNA in HEK293 cells. HEK293 cells were treated with various doses of H 2 O 2 for 10 min and harvested 1 h later. (A) Cytoplasmic extracts were incubated with biotinylated 31-mer wild-type or A4

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Incubation

    The NRF2 5′ UTR contains the G-quadruplex sequence and structure. (A) The NRF2 5′-UTR sequence is shown, with the putative G-quadruplex consensus sequence underlined. (B) A 31-mer RNA oligonucleotide (5 μM) synthesized from the

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: The NRF2 5′ UTR contains the G-quadruplex sequence and structure. (A) The NRF2 5′-UTR sequence is shown, with the putative G-quadruplex consensus sequence underlined. (B) A 31-mer RNA oligonucleotide (5 μM) synthesized from the

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Sequencing, Synthesized

    H 2 O 2 time-dependent increase of EF1a interactions with the NRF2 5′ UTR in cells. HEK293 cells were treated with 100 μM H 2 O 2 for 10 min and harvested at the indicated time points. (A) Cytoplasmic extracts were incubated with biotinylated

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: H 2 O 2 time-dependent increase of EF1a interactions with the NRF2 5′ UTR in cells. HEK293 cells were treated with 100 μM H 2 O 2 for 10 min and harvested at the indicated time points. (A) Cytoplasmic extracts were incubated with biotinylated

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Incubation

    H 2 O 2 treatment causes increased NRF2 protein levels in the absence of increased NRF2 mRNA levels. HEK293 cells were treated with various doses of H 2 O 2 (A) or 100 μM H 2 O 2 (B to D) for 10 min before harvesting 1 h later (A and D) or at the indicated

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: H 2 O 2 treatment causes increased NRF2 protein levels in the absence of increased NRF2 mRNA levels. HEK293 cells were treated with various doses of H 2 O 2 (A) or 100 μM H 2 O 2 (B to D) for 10 min before harvesting 1 h later (A and D) or at the indicated

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques:

    EF1a-dependent Nrf2 protein translation. HEK293 cells were transfected with either EF1a siRNA (siEF1) or negative-control siRNA (siNeg) without (A) or with (B) the pRL-Nrf25′UTR-FL reporter construct. Forty-eight hours after transfection, cells

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: EF1a-dependent Nrf2 protein translation. HEK293 cells were transfected with either EF1a siRNA (siEF1) or negative-control siRNA (siNeg) without (A) or with (B) the pRL-Nrf25′UTR-FL reporter construct. Forty-eight hours after transfection, cells

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Transfection, Negative Control, Construct

    G-quadruplex structure in solution containing H 2 O 2 and Fe 2+ . A 31-mer RNA oligonucleotide synthesized from the DNA template containing the sequence spanning nt −198 to −168 of the NRF2 5′ UTR was diluted in a solution containing

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: G-quadruplex structure in solution containing H 2 O 2 and Fe 2+ . A 31-mer RNA oligonucleotide synthesized from the DNA template containing the sequence spanning nt −198 to −168 of the NRF2 5′ UTR was diluted in a solution containing

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Synthesized, Sequencing

    NRF2 5′-UTR G-quadruplex binds to EF1a protein. A biotinylated 31-mer RNA fragment from the region spanning nt −198 to −168 of the NRF2 5′ UTR (WT) or the A4 mutant (MUT) was used as a bait for isolation of binding proteins

    Journal: Molecular and Cellular Biology

    Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress

    doi: 10.1128/MCB.00122-16

    Figure Lengend Snippet: NRF2 5′-UTR G-quadruplex binds to EF1a protein. A biotinylated 31-mer RNA fragment from the region spanning nt −198 to −168 of the NRF2 5′ UTR (WT) or the A4 mutant (MUT) was used as a bait for isolation of binding proteins

    Article Snippet: Separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for blotting with antibodies against Nrf2 (EP1808Y monoclonal [Abcam, Cambridge, MA] and H-300 polyclonal [Santa Cruz Biotechnology, CA]), vinculin (ab11194 monoclonal; Abcam, Cambridge, MA), or EF1a (H-300, catalog number sc-28578; Santa Cruz Biotechnology, CA).

    Techniques: Mutagenesis, Isolation, Binding Assay

    Redox imbalance in nephritic kidneys from OPN −/− mice: Western blot analysis of Nox4 ( A and B ) and Nrf2 ( C ) shows increased expression of Nox4 and blunted expression of Nrf2. Representative Western blots are shown. Densitometry analyses

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Inflammation and Inflammatory Mediators in Kidney Disease: Different effects of global osteopontin and macrophage osteopontin in glomerular injury

    doi: 10.1152/ajprenal.00458.2017

    Figure Lengend Snippet: Redox imbalance in nephritic kidneys from OPN −/− mice: Western blot analysis of Nox4 ( A and B ) and Nrf2 ( C ) shows increased expression of Nox4 and blunted expression of Nrf2. Representative Western blots are shown. Densitometry analyses

    Article Snippet: The protein levels of NADPH oxidase (Nox) 4 and Nrf2 were analyzed by Western blot using anti-NOX4 Ab (Novus Cell Signaling Technology, Danvers, MA) and Nrf2 Ab (Abcam, Cambridge, MA).

    Techniques: Mouse Assay, Western Blot, Expressing

    Pre-treatment with tert-butylhydroquinone (tBHQ) restores the total nuclear factor erythroid 2-related factor 2 (Nrf2) protein level and nuclear accumulation which are inhibited by ethanol (EtOH) in H9c2 cells. Western blot analysis was performed to analyze the level of Nrf2 protein. Cell lysates were prepared from H9c2 cells cultured in control medium (control), exposed to 200 mM EtOH or 5 µ M tBHQ (tBHQ) alone, or exposed to EtOH and pre-treated with tBHQ (EtOH + tBHQ). The data are expressed as fold change over control and represent the means ± SD of 3 separate experiments; * p

    Journal: International Journal of Molecular Medicine

    Article Title: Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes

    doi: 10.3892/ijmm.2016.2605

    Figure Lengend Snippet: Pre-treatment with tert-butylhydroquinone (tBHQ) restores the total nuclear factor erythroid 2-related factor 2 (Nrf2) protein level and nuclear accumulation which are inhibited by ethanol (EtOH) in H9c2 cells. Western blot analysis was performed to analyze the level of Nrf2 protein. Cell lysates were prepared from H9c2 cells cultured in control medium (control), exposed to 200 mM EtOH or 5 µ M tBHQ (tBHQ) alone, or exposed to EtOH and pre-treated with tBHQ (EtOH + tBHQ). The data are expressed as fold change over control and represent the means ± SD of 3 separate experiments; * p

    Article Snippet: The cells were then incubated overnight with primary antibodies against AC-tubulin (6-11B-1; ab24610) at 1:200 dilution and Nrf2 (both from Abcam) at a 1:200 dilution in a humidified chamber at 4°C.

    Techniques: Western Blot, Cell Culture

    The immunofluorescence assay evidenced that tert-butylhydroquinone (tBHQ) promotes the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in H9C2 cells. Nrf2 protein was visualized with a TRITC-labeled antibody, AC-tubulin was visualized with an FITC-labeled antibody, and the nuclear morphology was visualized with DAPI dye.

    Journal: International Journal of Molecular Medicine

    Article Title: Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes

    doi: 10.3892/ijmm.2016.2605

    Figure Lengend Snippet: The immunofluorescence assay evidenced that tert-butylhydroquinone (tBHQ) promotes the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in H9C2 cells. Nrf2 protein was visualized with a TRITC-labeled antibody, AC-tubulin was visualized with an FITC-labeled antibody, and the nuclear morphology was visualized with DAPI dye.

    Article Snippet: The cells were then incubated overnight with primary antibodies against AC-tubulin (6-11B-1; ab24610) at 1:200 dilution and Nrf2 (both from Abcam) at a 1:200 dilution in a humidified chamber at 4°C.

    Techniques: Immunofluorescence, Translocation Assay, Labeling

    Schematic representation of the working model In gastric cancer, TCF7L1 inhibits Keap1 expression, leading to enhanced NRF2 expression and constitutive activation of glycolysis and the antioxidant response, which ultimately contributes to uncontrolled proliferation of gastric cancer cells and poor prognosis in gastric cancer patients.

    Journal: Acta Biochimica et Biophysica Sinica

    Article Title: TCF7L1 indicates prognosis and promotes proliferation through activation of Keap1/NRF2 in gastric cancer

    doi: 10.1093/abbs/gmz015

    Figure Lengend Snippet: Schematic representation of the working model In gastric cancer, TCF7L1 inhibits Keap1 expression, leading to enhanced NRF2 expression and constitutive activation of glycolysis and the antioxidant response, which ultimately contributes to uncontrolled proliferation of gastric cancer cells and poor prognosis in gastric cancer patients.

    Article Snippet: Antibodies against TCF7L1 and NRF2 were purchased from Abcam (Cambridge, UK).

    Techniques: Expressing, Activation Assay

    TCF7L1 negatively regulates Keap1 expression in gastric cancer cells TCF7L1 could influence NRF2 in protein level. We hypothesized that this might due to the impact of TCF7L1 on Keap1 transcription, which could regulate NRF2 at the post-translational level. (A) Silencing of TCF7L1 increased Keap1 at mRNA level in AGS and MGC-803 cells. (B) Silencing of TCF7L1 expression increased Keap1 at protein level. (C,D) TCF7L1 could inhibit Keap1 promoter activity in a dose-dependent manner. (E) ChIP results validated that TCF7L1 could bind with the Keap1 promoter region.

    Journal: Acta Biochimica et Biophysica Sinica

    Article Title: TCF7L1 indicates prognosis and promotes proliferation through activation of Keap1/NRF2 in gastric cancer

    doi: 10.1093/abbs/gmz015

    Figure Lengend Snippet: TCF7L1 negatively regulates Keap1 expression in gastric cancer cells TCF7L1 could influence NRF2 in protein level. We hypothesized that this might due to the impact of TCF7L1 on Keap1 transcription, which could regulate NRF2 at the post-translational level. (A) Silencing of TCF7L1 increased Keap1 at mRNA level in AGS and MGC-803 cells. (B) Silencing of TCF7L1 expression increased Keap1 at protein level. (C,D) TCF7L1 could inhibit Keap1 promoter activity in a dose-dependent manner. (E) ChIP results validated that TCF7L1 could bind with the Keap1 promoter region.

    Article Snippet: Antibodies against TCF7L1 and NRF2 were purchased from Abcam (Cambridge, UK).

    Techniques: Expressing, Activity Assay, Chromatin Immunoprecipitation

    TCF7L1 positively regulates NRF2 at protein level and NRF2 downstream transcription program NRF2 is an important regulator of glycolysis and intracellular redox balance, thus we measured the impact of TCF7L1 on NRF2 expression. (A,B) Silencing of TCF7L1 expression had slight impact on NRF2 transcription. (C) NRF2 protein level was decreased in TCF7L1-silenced AGS and MGC-803 cells. (D,E) Silencing of TCF7L1 expression decreased the transcription of NRF2-targeted antioxidant genes, including GCLC , GCLM , HMOX , ME , TXNRD , and NQO1 . (F,G) NRF2 transcription activity was measured by ARE-luciferase activity assay. TCF7L1 increased ARE-luciferase activity in a dose-dependent manner.

    Journal: Acta Biochimica et Biophysica Sinica

    Article Title: TCF7L1 indicates prognosis and promotes proliferation through activation of Keap1/NRF2 in gastric cancer

    doi: 10.1093/abbs/gmz015

    Figure Lengend Snippet: TCF7L1 positively regulates NRF2 at protein level and NRF2 downstream transcription program NRF2 is an important regulator of glycolysis and intracellular redox balance, thus we measured the impact of TCF7L1 on NRF2 expression. (A,B) Silencing of TCF7L1 expression had slight impact on NRF2 transcription. (C) NRF2 protein level was decreased in TCF7L1-silenced AGS and MGC-803 cells. (D,E) Silencing of TCF7L1 expression decreased the transcription of NRF2-targeted antioxidant genes, including GCLC , GCLM , HMOX , ME , TXNRD , and NQO1 . (F,G) NRF2 transcription activity was measured by ARE-luciferase activity assay. TCF7L1 increased ARE-luciferase activity in a dose-dependent manner.

    Article Snippet: Antibodies against TCF7L1 and NRF2 were purchased from Abcam (Cambridge, UK).

    Techniques: Expressing, Activity Assay, Luciferase

    Andr inhibited hypoxia-induced oxidative stress via enhancing Nrf2/HO-1 pathway in H9C2 cells. H9C2 Cardiomyocytes were treatment with or without tri-gas incubator (Panasonic, Japan) and treated with different concentrations of Andr (0, 12.5, 25, or 50 µM). A and B . DCFH-DA staining (10μmol/L at 37°C for 20 min, ROS Assay Kit, Biyotime) and fluorescence intensity quantification were performed on H9C2 in vitro . C-G . Representative blots showed the expression of Keap1, Nrf2, HO-1 and HIF1α (C) and average fold-change (D-G) in the hearts (n=6 per groups). H . Immunofluorescence staining of Nrf2 in the indicated groups. I and J . Representative blots and histogram of Nrf2 expression in the n uclei of H9C2 (n =6 per groups). And the data are given as the mean ± SEM.∗ P

    Journal: International Journal of Biological Sciences

    Article Title: Andrographolide Protects Against Adverse Cardiac Remodeling After Myocardial Infarction through Enhancing Nrf2 Signaling Pathway

    doi: 10.7150/ijbs.37269

    Figure Lengend Snippet: Andr inhibited hypoxia-induced oxidative stress via enhancing Nrf2/HO-1 pathway in H9C2 cells. H9C2 Cardiomyocytes were treatment with or without tri-gas incubator (Panasonic, Japan) and treated with different concentrations of Andr (0, 12.5, 25, or 50 µM). A and B . DCFH-DA staining (10μmol/L at 37°C for 20 min, ROS Assay Kit, Biyotime) and fluorescence intensity quantification were performed on H9C2 in vitro . C-G . Representative blots showed the expression of Keap1, Nrf2, HO-1 and HIF1α (C) and average fold-change (D-G) in the hearts (n=6 per groups). H . Immunofluorescence staining of Nrf2 in the indicated groups. I and J . Representative blots and histogram of Nrf2 expression in the n uclei of H9C2 (n =6 per groups). And the data are given as the mean ± SEM.∗ P

    Article Snippet: For immunohistochemistry, the heart paraffin sections were heated using the pressure cooker for antigen retrieval and 8% goat serum was used to block nonspecific binding sites., incubated with anti-CD68 (ab125212, Abcam), anti-CD45 antibody (ab10558, Abcam), anti-Nrf2 antibody (ab137550, Abcam) and 4-hydroxynonenal (ab46545, Abcam) followed by incubation with goat anti-rabbit EnVisionTM+/ horseradish peroxidase (HRP) reagent, and stained using a DAB detection kit (Gene Tech, Shanghai, China).

    Techniques: Staining, ROS Assay, Fluorescence, In Vitro, Expressing, Immunofluorescence

    Andr suppressed oxidative stress after myocardial infarction by enhancing Nrf2/HO-1 pathway in mice. A-E . Representative Western blot analysis of Keap-1, Nrf2, HO-1 and HIF1α (A) and average fold-change (B-E) in the hearts (n=6 per groups). F and G . Immunohistochemical staining of Nrf2 in the indicated groups 3 weeks post-MI surgery or sham (F) and the Nrf2 positive cell for quantitative analysis (G) in mouse hearts. H and I . Representative blots and histogram of Nrf2 expression in the nuclei (n =6 per groups). J and K . Real-time PCR analysis of Nrf2 and HO-1. *P

    Journal: International Journal of Biological Sciences

    Article Title: Andrographolide Protects Against Adverse Cardiac Remodeling After Myocardial Infarction through Enhancing Nrf2 Signaling Pathway

    doi: 10.7150/ijbs.37269

    Figure Lengend Snippet: Andr suppressed oxidative stress after myocardial infarction by enhancing Nrf2/HO-1 pathway in mice. A-E . Representative Western blot analysis of Keap-1, Nrf2, HO-1 and HIF1α (A) and average fold-change (B-E) in the hearts (n=6 per groups). F and G . Immunohistochemical staining of Nrf2 in the indicated groups 3 weeks post-MI surgery or sham (F) and the Nrf2 positive cell for quantitative analysis (G) in mouse hearts. H and I . Representative blots and histogram of Nrf2 expression in the nuclei (n =6 per groups). J and K . Real-time PCR analysis of Nrf2 and HO-1. *P

    Article Snippet: For immunohistochemistry, the heart paraffin sections were heated using the pressure cooker for antigen retrieval and 8% goat serum was used to block nonspecific binding sites., incubated with anti-CD68 (ab125212, Abcam), anti-CD45 antibody (ab10558, Abcam), anti-Nrf2 antibody (ab137550, Abcam) and 4-hydroxynonenal (ab46545, Abcam) followed by incubation with goat anti-rabbit EnVisionTM+/ horseradish peroxidase (HRP) reagent, and stained using a DAB detection kit (Gene Tech, Shanghai, China).

    Techniques: Mouse Assay, Western Blot, Immunohistochemistry, Staining, Expressing, Real-time Polymerase Chain Reaction

    Nrf2 inhibition abolished the anti-oxidant effect of Andr in vitro . H9C2 Cardiomyocytes were transfected with siRNA for Nrf2 or Nrf2 inhibitor ML385 for 24h, followed by treatment with tri-gas incubator (Panasonic, Japan) or Andr for another 24h. A and B . DCFH-DA staining (10μmol/L at 37°C for 20 min, ROS Assay Kit, Biyotime) and fluorescence intensity quantification were performed on H9C2 in indicated condition in vitro . C-D . Representative blots and histogram of HO-1 expression in each group (n=6). ∗P

    Journal: International Journal of Biological Sciences

    Article Title: Andrographolide Protects Against Adverse Cardiac Remodeling After Myocardial Infarction through Enhancing Nrf2 Signaling Pathway

    doi: 10.7150/ijbs.37269

    Figure Lengend Snippet: Nrf2 inhibition abolished the anti-oxidant effect of Andr in vitro . H9C2 Cardiomyocytes were transfected with siRNA for Nrf2 or Nrf2 inhibitor ML385 for 24h, followed by treatment with tri-gas incubator (Panasonic, Japan) or Andr for another 24h. A and B . DCFH-DA staining (10μmol/L at 37°C for 20 min, ROS Assay Kit, Biyotime) and fluorescence intensity quantification were performed on H9C2 in indicated condition in vitro . C-D . Representative blots and histogram of HO-1 expression in each group (n=6). ∗P

    Article Snippet: For immunohistochemistry, the heart paraffin sections were heated using the pressure cooker for antigen retrieval and 8% goat serum was used to block nonspecific binding sites., incubated with anti-CD68 (ab125212, Abcam), anti-CD45 antibody (ab10558, Abcam), anti-Nrf2 antibody (ab137550, Abcam) and 4-hydroxynonenal (ab46545, Abcam) followed by incubation with goat anti-rabbit EnVisionTM+/ horseradish peroxidase (HRP) reagent, and stained using a DAB detection kit (Gene Tech, Shanghai, China).

    Techniques: Inhibition, In Vitro, Transfection, Staining, ROS Assay, Fluorescence, Expressing

    Effect of ferrostatin-1 on the expression of Nrf2 (A) and Gpx4 (B) after Glu treatment in HT-22 cells. Western blot data represent as the mean ± SD. ## P

    Journal: Neural Regeneration Research

    Article Title: Ferrostatin-1 protects HT-22 cells from oxidative toxicity

    doi: 10.4103/1673-5374.266060

    Figure Lengend Snippet: Effect of ferrostatin-1 on the expression of Nrf2 (A) and Gpx4 (B) after Glu treatment in HT-22 cells. Western blot data represent as the mean ± SD. ## P

    Article Snippet: After blocking with nonfat milk for 1 hour, nitrocellulose filter membranes were incubated with primary antibodies (rabbit anti-mouse Gpx4 antibody, 1:1000, ab125066, Abcam, Shanghai, China; rabbit anti-Nrf2 antibody, 1:1000, ab137550, Abcam) overnight at 4°C.

    Techniques: Expressing, Western Blot

    Effects of THC on Nrf2 activation and HO-1 expression. (A B) Phosphorylation and nuclear translocation of Nrf2. BV2 microglia cells were treated with THC (10-100 μM), and then Western blot analysis was performed. Total level of phosphorylated Nrf2 was determined (A) and nuclear translocation of Nrf2 was examined in nuclear and cytosol fractions (B). (C, D) HO-1 protein and mRNA expression. HO-1 protein level (C) was determined by immunoblotting after THC treatment. After 6 hr of THC treatment, the total RNA was isolated, HO-1 mRNA level (D) was determined by RT-PCR. β-Actin was used as an internal control. Quantitative analysis was carried out using densitometric analysis. Images are representative of three independent experiments that shows similar results. * p

    Journal: Biomolecules & Therapeutics

    Article Title: 3,4,5-Trihydroxycinnamic Acid Inhibits Lipopolysaccharide-Induced Inflammatory Response through the Activation of Nrf2 Pathway in BV2 Microglial Cells

    doi: 10.4062/biomolther.2012.091

    Figure Lengend Snippet: Effects of THC on Nrf2 activation and HO-1 expression. (A B) Phosphorylation and nuclear translocation of Nrf2. BV2 microglia cells were treated with THC (10-100 μM), and then Western blot analysis was performed. Total level of phosphorylated Nrf2 was determined (A) and nuclear translocation of Nrf2 was examined in nuclear and cytosol fractions (B). (C, D) HO-1 protein and mRNA expression. HO-1 protein level (C) was determined by immunoblotting after THC treatment. After 6 hr of THC treatment, the total RNA was isolated, HO-1 mRNA level (D) was determined by RT-PCR. β-Actin was used as an internal control. Quantitative analysis was carried out using densitometric analysis. Images are representative of three independent experiments that shows similar results. * p

    Article Snippet: Specific antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK 1/2, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK (1:1,000, Cell signaling), poly (ADP-ribose) polymerase (PARP) (1:1,000), Nrf2 (1:1,000, Santa Cruz), p-Nrf2 (1:1,000, Abcam), HO-1 (1:1,000; Abcam), MCP (1;1,000; Abcam) and β-actin (1; 2,500; Sigma) were diluted in 5% skim milk.

    Techniques: Activation Assay, Expressing, Translocation Assay, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

    THC-induced Nrf2 phosphorylation was mediated by p-38 MAPK. BV2 microglial cells were stimulated with 200 ng/ml LPS in the absence or presence of THC. (A) Western blot analysis was then performed to evaluate the activation of MAP kinases signaling pathways (top: representative image, bottom: quantitative analysis). Phosphorylation of p38 was increased in a concentration-dependent manner, but phosphorylation of ERK and JNK was decreased with THC treatment, suggesting that p-38 might play an important role in the THC-induced Nrf2 phosphorylation. (B) BV2 microglia cells were pretreated with the p38 inhibitor, SB203580 (5 μM), for 1 hr prior to THC treatment. THC-induced Nrf2 phosphorylation was blocked via p38 inhibition (top: representative image, bottom: quantitative analysis), indicating that p38 is responsible for the THC-induced phosphorylation of Nrf2. β-Actin was used as an internal control. Images are representative of three independent experiments that shows similar results. Quantitative analysis was carried out using densitometric analysis. * p

    Journal: Biomolecules & Therapeutics

    Article Title: 3,4,5-Trihydroxycinnamic Acid Inhibits Lipopolysaccharide-Induced Inflammatory Response through the Activation of Nrf2 Pathway in BV2 Microglial Cells

    doi: 10.4062/biomolther.2012.091

    Figure Lengend Snippet: THC-induced Nrf2 phosphorylation was mediated by p-38 MAPK. BV2 microglial cells were stimulated with 200 ng/ml LPS in the absence or presence of THC. (A) Western blot analysis was then performed to evaluate the activation of MAP kinases signaling pathways (top: representative image, bottom: quantitative analysis). Phosphorylation of p38 was increased in a concentration-dependent manner, but phosphorylation of ERK and JNK was decreased with THC treatment, suggesting that p-38 might play an important role in the THC-induced Nrf2 phosphorylation. (B) BV2 microglia cells were pretreated with the p38 inhibitor, SB203580 (5 μM), for 1 hr prior to THC treatment. THC-induced Nrf2 phosphorylation was blocked via p38 inhibition (top: representative image, bottom: quantitative analysis), indicating that p38 is responsible for the THC-induced phosphorylation of Nrf2. β-Actin was used as an internal control. Images are representative of three independent experiments that shows similar results. Quantitative analysis was carried out using densitometric analysis. * p

    Article Snippet: Specific antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK 1/2, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK (1:1,000, Cell signaling), poly (ADP-ribose) polymerase (PARP) (1:1,000), Nrf2 (1:1,000, Santa Cruz), p-Nrf2 (1:1,000, Abcam), HO-1 (1:1,000; Abcam), MCP (1;1,000; Abcam) and β-actin (1; 2,500; Sigma) were diluted in 5% skim milk.

    Techniques: Western Blot, Activation Assay, Concentration Assay, Inhibition

    Augmentation of GSNO controls Ch-CS-induced nitrosative/oxidative stress. (A, B) Immunostaining of longitudinal lung sections from C57BL/6 mice exposed to RA or Ch-CS exposure (18 weeks, 5 h/day, 5 days/week) and/or treated with vehicle control (PBS), GSNO, or N6022 (4 mg/kg body weight, i.t., “three” total doses with a gap of 3 days) demonstrates that Ch-CS-mediated increase in iNOS and 3-nitrotyrosine (nitrosative/oxidative stress markers) expression (high-magnification images shown as insets ) is significantly diminished by restoring intracellular GSNO levels by GSNO or GSNOR inhibitor (N6022). (C) The longitudinal lung sections described in (A) were immunostained with p-Nrf2 (antioxidant response marker) and the data indicate that Ch-CS-induced decrease in nuclear localization of p-Nrf2 ( yellow arrows , insets ) is significantly restored by GSNO or GSNOR inhibitor (N6022)-mediated augmentation of GSNO levels. The data also demonstrate that p-Nrf2 accumulates in the perinuclear spaces ( red arrows ) in Ch-CS-exposed mice lungs, which can be rescued by GSNO augmentation. (D–F) Data analysis represents average of four replicates shown as mean ± SEM [* p

    Journal: Antioxidants & Redox Signaling

    Article Title: Augmentation of S-Nitrosoglutathione Controls Cigarette Smoke-Induced Inflammatory–Oxidative Stress and Chronic Obstructive Pulmonary Disease-Emphysema Pathogenesis by Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function

    doi: 10.1089/ars.2016.6895

    Figure Lengend Snippet: Augmentation of GSNO controls Ch-CS-induced nitrosative/oxidative stress. (A, B) Immunostaining of longitudinal lung sections from C57BL/6 mice exposed to RA or Ch-CS exposure (18 weeks, 5 h/day, 5 days/week) and/or treated with vehicle control (PBS), GSNO, or N6022 (4 mg/kg body weight, i.t., “three” total doses with a gap of 3 days) demonstrates that Ch-CS-mediated increase in iNOS and 3-nitrotyrosine (nitrosative/oxidative stress markers) expression (high-magnification images shown as insets ) is significantly diminished by restoring intracellular GSNO levels by GSNO or GSNOR inhibitor (N6022). (C) The longitudinal lung sections described in (A) were immunostained with p-Nrf2 (antioxidant response marker) and the data indicate that Ch-CS-induced decrease in nuclear localization of p-Nrf2 ( yellow arrows , insets ) is significantly restored by GSNO or GSNOR inhibitor (N6022)-mediated augmentation of GSNO levels. The data also demonstrate that p-Nrf2 accumulates in the perinuclear spaces ( red arrows ) in Ch-CS-exposed mice lungs, which can be rescued by GSNO augmentation. (D–F) Data analysis represents average of four replicates shown as mean ± SEM [* p

    Article Snippet: The primary antibodies used were CFTR (rabbit polyclonal; Santa Cruz Biotechnology), p62 (rabbit polyclonal; Santa Cruz Biotechnology), iNOS (rabbit polyclonal; Santa Cruz Biotechnology), 3-nitrotyrosine (rabbit polyclonal; Santa Cruz Biotechnology), p-Nrf2 (rabbit monoclonal; Abcam), or Ub (mouse polyclonal; Santa Cruz Biotechnology).

    Techniques: Immunostaining, Mouse Assay, Expressing, Marker

    Schematic depicting neuroprotective roles of BRCA1 towards I/R injury. Excessive ROS production damages macromolecule DNA and lipids. BRCA1 binds directly to NRF2 via its BRCT domain and facilitates NRF2-mediated antioxidant response. Then the downstream ARE genes, including HO-1, NQO1, GCLC, GPX4 and SOD2 are induced, which reduces ROS production and attenuates lipid peroxidation. Meanwhile, BRCA1 promotes DSBs repair through up-regulating Ku70/Ku80 in NHEJ pathway. As a result, neuronal cell death is inhibited.

    Journal: Redox Biology

    Article Title: Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway

    doi: 10.1016/j.redox.2018.06.012

    Figure Lengend Snippet: Schematic depicting neuroprotective roles of BRCA1 towards I/R injury. Excessive ROS production damages macromolecule DNA and lipids. BRCA1 binds directly to NRF2 via its BRCT domain and facilitates NRF2-mediated antioxidant response. Then the downstream ARE genes, including HO-1, NQO1, GCLC, GPX4 and SOD2 are induced, which reduces ROS production and attenuates lipid peroxidation. Meanwhile, BRCA1 promotes DSBs repair through up-regulating Ku70/Ku80 in NHEJ pathway. As a result, neuronal cell death is inhibited.

    Article Snippet: Membranes were blocked with 3% bovine serum albumin and 5% fat-free milk at room temperature for 1.5 h. Then membranes were incubated with primary antibodies against BRCA1 (1:1000, Abcam, UK), p53 (1:1000, Abcam, UK), Bcl-2 (1:1000, Abcam, UK), Bax (1:1000, Cell Signaling technology, USA), Cleaved Caspase-3 (1:1000, Cell Signaling technology, USA), γH2A.X (1:1000, Millipore, USA), RAD51 (1:5000, Millipore, USA), Ku70/80 (1:1000, Abcam, UK), pDNA-PKcs (1:1000, Abcam, UK), DNA-PKcs (1:1000, Abcam, UK), NRF2 (1:1000, Abcam, UK), NQO1 (1:1000, Abcam, UK), HO-1 (1:200, Santa Cruz, USA), GPX4 (1:1000, Abcam, UK), PSD95 (1:1000, Abcam, UK), CaMKII (1:1000, Abcam, UK), Synapsin I (1:200, Santa Cruz, USA), Synaptophysin (1:1000, Abcam, UK), GST (1:50000, Cell Signaling technology, USA), histone H3 (1:3000, Cell Signaling technology, USA) and β-actin (1:4000, Cell Signaling technology, USA) overnight at 4 °C, followed by incubated with HRP-conjugated secondary antibody at room temperature.

    Techniques: Non-Homologous End Joining

    BRCA1 provoked NRF2-mediated antioxidative pathway. (A) BRCA1 binds to NRF2. The brain lysates were immunoprecipitated with anti-BRCA1 (left) or anti-NRF2 (right) antibodies. Then total lysates and immunoprecipitates were analyzed by immunoblotting with anti-BRCA1 and anti-NRF2. n = 5. (B) Schematic representation of BRCA1 fusion proteins. Interaction was detected between BRCT domians (aa 1591–1784) and NRF2. (C) Western blots showing the protein levels of total, cytosolic and nuclear NRF2, and quantification analysis of total and nuclear NRF2. n = 6. (D) mRNA levels of NRF2 in the indicated groups, n = 6. (E) Dual-luciferase reporter assay of BRCA1 and NRF2 XRE, n = 5. (F) mRNA levels of HO-1, NQO1, GPX4, GCLC, GCLM, SOD1 and SOD2. n = 6. (G) Immunoblots and quantitative analysis of total HO-1, NQO1 and GPX4. n = 6. (H) Schematic diagram of NRF2-mediated antioxidative pathway triggered by BRCA1. Data are expressed as mean ± SD; † † † P

    Journal: Redox Biology

    Article Title: Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway

    doi: 10.1016/j.redox.2018.06.012

    Figure Lengend Snippet: BRCA1 provoked NRF2-mediated antioxidative pathway. (A) BRCA1 binds to NRF2. The brain lysates were immunoprecipitated with anti-BRCA1 (left) or anti-NRF2 (right) antibodies. Then total lysates and immunoprecipitates were analyzed by immunoblotting with anti-BRCA1 and anti-NRF2. n = 5. (B) Schematic representation of BRCA1 fusion proteins. Interaction was detected between BRCT domians (aa 1591–1784) and NRF2. (C) Western blots showing the protein levels of total, cytosolic and nuclear NRF2, and quantification analysis of total and nuclear NRF2. n = 6. (D) mRNA levels of NRF2 in the indicated groups, n = 6. (E) Dual-luciferase reporter assay of BRCA1 and NRF2 XRE, n = 5. (F) mRNA levels of HO-1, NQO1, GPX4, GCLC, GCLM, SOD1 and SOD2. n = 6. (G) Immunoblots and quantitative analysis of total HO-1, NQO1 and GPX4. n = 6. (H) Schematic diagram of NRF2-mediated antioxidative pathway triggered by BRCA1. Data are expressed as mean ± SD; † † † P

    Article Snippet: Membranes were blocked with 3% bovine serum albumin and 5% fat-free milk at room temperature for 1.5 h. Then membranes were incubated with primary antibodies against BRCA1 (1:1000, Abcam, UK), p53 (1:1000, Abcam, UK), Bcl-2 (1:1000, Abcam, UK), Bax (1:1000, Cell Signaling technology, USA), Cleaved Caspase-3 (1:1000, Cell Signaling technology, USA), γH2A.X (1:1000, Millipore, USA), RAD51 (1:5000, Millipore, USA), Ku70/80 (1:1000, Abcam, UK), pDNA-PKcs (1:1000, Abcam, UK), DNA-PKcs (1:1000, Abcam, UK), NRF2 (1:1000, Abcam, UK), NQO1 (1:1000, Abcam, UK), HO-1 (1:200, Santa Cruz, USA), GPX4 (1:1000, Abcam, UK), PSD95 (1:1000, Abcam, UK), CaMKII (1:1000, Abcam, UK), Synapsin I (1:200, Santa Cruz, USA), Synaptophysin (1:1000, Abcam, UK), GST (1:50000, Cell Signaling technology, USA), histone H3 (1:3000, Cell Signaling technology, USA) and β-actin (1:4000, Cell Signaling technology, USA) overnight at 4 °C, followed by incubated with HRP-conjugated secondary antibody at room temperature.

    Techniques: Immunoprecipitation, Western Blot, Luciferase, Reporter Assay

    BRCA1 provoked NRF2-mediated antioxidative pathway. (A) BRCA1 binds to NRF2. The brain lysates were immunoprecipitated with anti-BRCA1 (left) or anti-NRF2 (right) antibodies. Then total lysates and immunoprecipitates were analyzed by immunoblotting with anti-BRCA1 and anti-NRF2. n = 5. (B) Schematic representation of BRCA1 fusion proteins. Interaction was detected between BRCT domians (aa 1591–1784) and NRF2. (C) Western blots showing the protein levels of total, cytosolic and nuclear NRF2, and quantification analysis of total and nuclear NRF2. n = 6. (D) mRNA levels of NRF2 in the indicated groups, n = 6. (E) Dual-luciferase reporter assay of BRCA1 and NRF2 XRE, n = 5. (F) mRNA levels of HO-1, NQO1, GPX4, GCLC, GCLM, SOD1 and SOD2. n = 6. (G) Immunoblots and quantitative analysis of total HO-1, NQO1 and GPX4. n = 6. (H) Schematic diagram of NRF2-mediated antioxidative pathway triggered by BRCA1. Data are expressed as mean ± SD; † † † P

    Journal: Redox Biology

    Article Title: Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway

    doi: 10.1016/j.redox.2018.06.012

    Figure Lengend Snippet: BRCA1 provoked NRF2-mediated antioxidative pathway. (A) BRCA1 binds to NRF2. The brain lysates were immunoprecipitated with anti-BRCA1 (left) or anti-NRF2 (right) antibodies. Then total lysates and immunoprecipitates were analyzed by immunoblotting with anti-BRCA1 and anti-NRF2. n = 5. (B) Schematic representation of BRCA1 fusion proteins. Interaction was detected between BRCT domians (aa 1591–1784) and NRF2. (C) Western blots showing the protein levels of total, cytosolic and nuclear NRF2, and quantification analysis of total and nuclear NRF2. n = 6. (D) mRNA levels of NRF2 in the indicated groups, n = 6. (E) Dual-luciferase reporter assay of BRCA1 and NRF2 XRE, n = 5. (F) mRNA levels of HO-1, NQO1, GPX4, GCLC, GCLM, SOD1 and SOD2. n = 6. (G) Immunoblots and quantitative analysis of total HO-1, NQO1 and GPX4. n = 6. (H) Schematic diagram of NRF2-mediated antioxidative pathway triggered by BRCA1. Data are expressed as mean ± SD; † † † P

    Article Snippet: Membranes were blocked with 3% bovine serum albumin and 5% fat-free milk at room temperature for 1.5 h. Then membranes were incubated with primary antibodies against BRCA1 (1:1000, Abcam, UK), p53 (1:1000, Abcam, UK), Bcl-2 (1:1000, Abcam, UK), Bax (1:1000, Cell Signaling technology, USA), Cleaved Caspase-3 (1:1000, Cell Signaling technology, USA), γH2A.X (1:1000, Millipore, USA), RAD51 (1:5000, Millipore, USA), Ku70/80 (1:1000, Abcam, UK), pDNA-PKcs (1:1000, Abcam, UK), DNA-PKcs (1:1000, Abcam, UK), NRF2 (1:1000, Abcam, UK), NQO1 (1:1000, Abcam, UK), HO-1 (1:200, Santa Cruz, USA), GPX4 (1:1000, Abcam, UK), PSD95 (1:1000, Abcam, UK), CaMKII (1:1000, Abcam, UK), Synapsin I (1:200, Santa Cruz, USA), Synaptophysin (1:1000, Abcam, UK), GST (1:50000, Cell Signaling technology, USA), histone H3 (1:3000, Cell Signaling technology, USA) and β-actin (1:4000, Cell Signaling technology, USA) overnight at 4 °C, followed by incubated with HRP-conjugated secondary antibody at room temperature.

    Techniques: Immunoprecipitation, Western Blot, Luciferase, Reporter Assay

    Schematic depicting neuroprotective roles of BRCA1 towards I/R injury. Excessive ROS production damages macromolecule DNA and lipids. BRCA1 binds directly to NRF2 via its BRCT domain and facilitates NRF2-mediated antioxidant response. Then the downstream ARE genes, including HO-1, NQO1, GCLC, GPX4 and SOD2 are induced, which reduces ROS production and attenuates lipid peroxidation. Meanwhile, BRCA1 promotes DSBs repair through up-regulating Ku70/Ku80 in NHEJ pathway. As a result, neuronal cell death is inhibited.

    Journal: Redox Biology

    Article Title: Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway

    doi: 10.1016/j.redox.2018.06.012

    Figure Lengend Snippet: Schematic depicting neuroprotective roles of BRCA1 towards I/R injury. Excessive ROS production damages macromolecule DNA and lipids. BRCA1 binds directly to NRF2 via its BRCT domain and facilitates NRF2-mediated antioxidant response. Then the downstream ARE genes, including HO-1, NQO1, GCLC, GPX4 and SOD2 are induced, which reduces ROS production and attenuates lipid peroxidation. Meanwhile, BRCA1 promotes DSBs repair through up-regulating Ku70/Ku80 in NHEJ pathway. As a result, neuronal cell death is inhibited.

    Article Snippet: Membranes were blocked with 3% bovine serum albumin and 5% fat-free milk at room temperature for 1.5 h. Then membranes were incubated with primary antibodies against BRCA1 (1:1000, Abcam, UK), p53 (1:1000, Abcam, UK), Bcl-2 (1:1000, Abcam, UK), Bax (1:1000, Cell Signaling technology, USA), Cleaved Caspase-3 (1:1000, Cell Signaling technology, USA), γH2A.X (1:1000, Millipore, USA), RAD51 (1:5000, Millipore, USA), Ku70/80 (1:1000, Abcam, UK), pDNA-PKcs (1:1000, Abcam, UK), DNA-PKcs (1:1000, Abcam, UK), NRF2 (1:1000, Abcam, UK), NQO1 (1:1000, Abcam, UK), HO-1 (1:200, Santa Cruz, USA), GPX4 (1:1000, Abcam, UK), PSD95 (1:1000, Abcam, UK), CaMKII (1:1000, Abcam, UK), Synapsin I (1:200, Santa Cruz, USA), Synaptophysin (1:1000, Abcam, UK), GST (1:50000, Cell Signaling technology, USA), histone H3 (1:3000, Cell Signaling technology, USA) and β-actin (1:4000, Cell Signaling technology, USA) overnight at 4 °C, followed by incubated with HRP-conjugated secondary antibody at room temperature.

    Techniques: Non-Homologous End Joining

    Expression of Nrf2, Maf G, and Nrf2-targert genes. ( A ) Immunoblot analyses of the proteins related to antioxidation and detoxification, Nrf2, phosphorylated Nrf2 (p-Nrf2), and Maf G for Huh7.5 and HPI cells. Fold expression of transcript (HPI/Huh7.5) in corresponding genes was shown in the right according to the expression array data. ( B ) Immunoblot analysis of p-Nrf2 in cytosol and nucleus fractions from the cells used in (A). GAPDH and Lamin A were used as a marker protein for cytosol and nucleus, respectively. ( C ) Immunoblot analyses of the HCV core and NS5A proteins, Nrf2-trarget genes, Nrf2, p-Nrf2 and Maf G for Huh7.5 and HPI cells, CuHuh7.5 cells (Huh7.5 cells simply treated with cyclosporine) and CuHPI cells (HPI cells, from which HCV was eliminated with cyclosporine). ( D ) Immunoblot analysis of p-Nrf2 in their cytosol and nucleus fractions from the cells used in (C).

    Journal: PLoS ONE

    Article Title: Prominent Steatosis with Hypermetabolism of the Cell Line Permissive for Years of Infection with Hepatitis C Virus

    doi: 10.1371/journal.pone.0094460

    Figure Lengend Snippet: Expression of Nrf2, Maf G, and Nrf2-targert genes. ( A ) Immunoblot analyses of the proteins related to antioxidation and detoxification, Nrf2, phosphorylated Nrf2 (p-Nrf2), and Maf G for Huh7.5 and HPI cells. Fold expression of transcript (HPI/Huh7.5) in corresponding genes was shown in the right according to the expression array data. ( B ) Immunoblot analysis of p-Nrf2 in cytosol and nucleus fractions from the cells used in (A). GAPDH and Lamin A were used as a marker protein for cytosol and nucleus, respectively. ( C ) Immunoblot analyses of the HCV core and NS5A proteins, Nrf2-trarget genes, Nrf2, p-Nrf2 and Maf G for Huh7.5 and HPI cells, CuHuh7.5 cells (Huh7.5 cells simply treated with cyclosporine) and CuHPI cells (HPI cells, from which HCV was eliminated with cyclosporine). ( D ) Immunoblot analysis of p-Nrf2 in their cytosol and nucleus fractions from the cells used in (C).

    Article Snippet: Primary and Secondary Antibodies for Immunofluorescence Stain and Immunoblot Analysis Primary antibodies were for HCV proteins: core (Institute of immunology), NS5A (Antiprot), NS3 (Antiprot), NS5A (Virogen), and NS5B (Antiprot), beta-Actin (Abcam), ACYL (Cell signaling), HMGCR (Atlas antibodies), DHCR7 (Abcam), ACACA (Santa Cruz), ELOVL5 (Novusbio), GPAM (Abcam), MTTP (Abcam), PNPLA3 (Abcam), GCK (Santa Cruz), GAPDH (Abcam), G6PD (Santa Cruz), PPAT (Proteintech), MTHFD2 (Proteintech), PSAT1 (Santa Cruz), SHMT1 (Abcam), G6PC3 (Santa Cruz), DLAT (Santa Cruz), IDH3G (Santa Cruz), ASNS (Santa Cruz), ME1 (Abcam), PCK2 (Abcam), GPT2 (Santa Cruz), NQO1 (Proteintech), GCLC (Abnova), Nrf2 (Santa Cruz), phosphorylated-Nrf2 (Abcam), Maf-G (Abcam), Lamin A (Santa Cruz).

    Techniques: Expressing, Marker

    Knockdown of Nrf2 reduced lipid droplets and HCV in HPI cell. For knockdown of Nrf2, HPI cells were transfected with siRNA for Nrf2 or negative control siRNA (Ct) three times and were analyzed 2 days after the last transfection. ( A ) Immunoblot analysis of the protein for Nrf2, Nrf2-target genes and HCV (core and NS5A) was performed. ( B ) RT-PCRs were performed for three parts of HCV (5′UTR-NS2, NS3-NS4B and NS5A-NS5B) covering the full genome of HCV. Number in parenthesis indicates HCV nucleotide position amplified by RT-PCR. The most left lane of each panel represents size maker (2, 3, 4, 5, 6, 8 and 10 kb) for HCV and that for β-actin (0.2, 0.3 and 0.4 kb). ( C ) Fluorescence staining for LDs and simultaneous immunofluorescence staining for HCV core and NS5A proteins (four left panels and four right panels, respectively) were performed. Nuclei were counterstained with DAPI. ( D ) Intracellular lipid contents were determined in triplicate. Values were corrected by the protein concentration and statistically evaluated by Student's t- test indicating a significance of P

    Journal: PLoS ONE

    Article Title: Prominent Steatosis with Hypermetabolism of the Cell Line Permissive for Years of Infection with Hepatitis C Virus

    doi: 10.1371/journal.pone.0094460

    Figure Lengend Snippet: Knockdown of Nrf2 reduced lipid droplets and HCV in HPI cell. For knockdown of Nrf2, HPI cells were transfected with siRNA for Nrf2 or negative control siRNA (Ct) three times and were analyzed 2 days after the last transfection. ( A ) Immunoblot analysis of the protein for Nrf2, Nrf2-target genes and HCV (core and NS5A) was performed. ( B ) RT-PCRs were performed for three parts of HCV (5′UTR-NS2, NS3-NS4B and NS5A-NS5B) covering the full genome of HCV. Number in parenthesis indicates HCV nucleotide position amplified by RT-PCR. The most left lane of each panel represents size maker (2, 3, 4, 5, 6, 8 and 10 kb) for HCV and that for β-actin (0.2, 0.3 and 0.4 kb). ( C ) Fluorescence staining for LDs and simultaneous immunofluorescence staining for HCV core and NS5A proteins (four left panels and four right panels, respectively) were performed. Nuclei were counterstained with DAPI. ( D ) Intracellular lipid contents were determined in triplicate. Values were corrected by the protein concentration and statistically evaluated by Student's t- test indicating a significance of P

    Article Snippet: Primary and Secondary Antibodies for Immunofluorescence Stain and Immunoblot Analysis Primary antibodies were for HCV proteins: core (Institute of immunology), NS5A (Antiprot), NS3 (Antiprot), NS5A (Virogen), and NS5B (Antiprot), beta-Actin (Abcam), ACYL (Cell signaling), HMGCR (Atlas antibodies), DHCR7 (Abcam), ACACA (Santa Cruz), ELOVL5 (Novusbio), GPAM (Abcam), MTTP (Abcam), PNPLA3 (Abcam), GCK (Santa Cruz), GAPDH (Abcam), G6PD (Santa Cruz), PPAT (Proteintech), MTHFD2 (Proteintech), PSAT1 (Santa Cruz), SHMT1 (Abcam), G6PC3 (Santa Cruz), DLAT (Santa Cruz), IDH3G (Santa Cruz), ASNS (Santa Cruz), ME1 (Abcam), PCK2 (Abcam), GPT2 (Santa Cruz), NQO1 (Proteintech), GCLC (Abnova), Nrf2 (Santa Cruz), phosphorylated-Nrf2 (Abcam), Maf-G (Abcam), Lamin A (Santa Cruz).

    Techniques: Transfection, Negative Control, Amplification, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Staining, Immunofluorescence, Protein Concentration

    Rescue patterns of suppressed osteogenic potential upon the removal of OTA or t-BHQ, and the association with NRF2 nuclear localization and stemness genes. ( a ) EP MSCs (8 × 10 4 cells per well in 12-well plates) treated with EtOH or OTA (10 μ M) were incubated in osteogenic medium for 10 days. In addition, OTA application was stopped on day 7 of osteogenic differentiation in another group. Alizarin red S staining was performed to detect mineral deposition at days 3, 7, and 10. For quantitative analysis, absorbance was measured at 595 nm following destaining with 10% cetylpyridinium for 30 min. * P

    Journal: Cell Death & Disease

    Article Title: Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    doi: 10.1038/cddis.2016.3

    Figure Lengend Snippet: Rescue patterns of suppressed osteogenic potential upon the removal of OTA or t-BHQ, and the association with NRF2 nuclear localization and stemness genes. ( a ) EP MSCs (8 × 10 4 cells per well in 12-well plates) treated with EtOH or OTA (10 μ M) were incubated in osteogenic medium for 10 days. In addition, OTA application was stopped on day 7 of osteogenic differentiation in another group. Alizarin red S staining was performed to detect mineral deposition at days 3, 7, and 10. For quantitative analysis, absorbance was measured at 595 nm following destaining with 10% cetylpyridinium for 30 min. * P

    Article Snippet: Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology).

    Techniques: Incubation, Staining

    NRF2-mediated SIRT1 regulation occurs in a p53-dependent manner in EP MSCs. ( a ) EP MSCs were incubated in basal growth medium (DMEM-LG containing 10% FBS) in the presence of EtOH, 1 μ M OTA, and 10 μ M OTA for 16 h. The mRNA expression levels of HO-1 , NQO-1 , and SIRT1 were also analyzed by qRT-PCR. * P

    Journal: Cell Death & Disease

    Article Title: Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    doi: 10.1038/cddis.2016.3

    Figure Lengend Snippet: NRF2-mediated SIRT1 regulation occurs in a p53-dependent manner in EP MSCs. ( a ) EP MSCs were incubated in basal growth medium (DMEM-LG containing 10% FBS) in the presence of EtOH, 1 μ M OTA, and 10 μ M OTA for 16 h. The mRNA expression levels of HO-1 , NQO-1 , and SIRT1 were also analyzed by qRT-PCR. * P

    Article Snippet: Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology).

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    OTA induces nuclear export of NRF2 and decreases the self-renewal capacity and osteogenic differentiation in EP MSCs. ( a ) EP-MSCs were incubated in basal growth medium (DMEM-LG containing 10% FBS) in the presence of EtOH or OTA (10 μ M) for 16 h. The cell lysates were prepared for EtOH-treated or OTA-treated EP MSCs, and then fractionated into nuclear and cytosolic extracts. The protein levels of NRF2 and phosphorylated NRF2 were analyzed by western blot analysis for EtOH-treated or OTA-treated EP MSCs. The protein level of LDH was used as a loading control for cytosolic extracts, and the protein level of LAMIN-B was used as a loading control for nuclear extracts. The expression level of β -CATENIN was also used as a loading control for both cytosolic and nuclear extracts. EtOH was used as a vehicle for OTA. ( b ) Immunofluorescence was performed to observe the nuclear and cytosolic localization of NRF2 in the EtOH-treated or OTA (10 μ M)-treated EP MSCs. The nuclei were stained with DAPI, and NRF2 was stained with Alexa Fluor 568 (Yellow)-conjugated secondary antibody. The images were obtained using confocal microscopy. Scale bar=100 μ m. ( c ) The mRNA expression levels of HO-1 and NQO-1 were also analyzed by qRT-PCR. * P

    Journal: Cell Death & Disease

    Article Title: Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    doi: 10.1038/cddis.2016.3

    Figure Lengend Snippet: OTA induces nuclear export of NRF2 and decreases the self-renewal capacity and osteogenic differentiation in EP MSCs. ( a ) EP-MSCs were incubated in basal growth medium (DMEM-LG containing 10% FBS) in the presence of EtOH or OTA (10 μ M) for 16 h. The cell lysates were prepared for EtOH-treated or OTA-treated EP MSCs, and then fractionated into nuclear and cytosolic extracts. The protein levels of NRF2 and phosphorylated NRF2 were analyzed by western blot analysis for EtOH-treated or OTA-treated EP MSCs. The protein level of LDH was used as a loading control for cytosolic extracts, and the protein level of LAMIN-B was used as a loading control for nuclear extracts. The expression level of β -CATENIN was also used as a loading control for both cytosolic and nuclear extracts. EtOH was used as a vehicle for OTA. ( b ) Immunofluorescence was performed to observe the nuclear and cytosolic localization of NRF2 in the EtOH-treated or OTA (10 μ M)-treated EP MSCs. The nuclei were stained with DAPI, and NRF2 was stained with Alexa Fluor 568 (Yellow)-conjugated secondary antibody. The images were obtained using confocal microscopy. Scale bar=100 μ m. ( c ) The mRNA expression levels of HO-1 and NQO-1 were also analyzed by qRT-PCR. * P

    Article Snippet: Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology).

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

    NRF2 activation by t-BHQ enhances the self-renewal capacity and osteogenic potential of LP MSCs. ( a ) LP MSCs were grown in basal growth medium (DMEM-LG containing 10% FBS) in the presence of t-BHQ (10 μ M) for 7 days. The mRNA expression levels of HO-1 , NQO-1 , ( b ) SIRT1 , and p53 were also analyzed by qRT-PCR. * P

    Journal: Cell Death & Disease

    Article Title: Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    doi: 10.1038/cddis.2016.3

    Figure Lengend Snippet: NRF2 activation by t-BHQ enhances the self-renewal capacity and osteogenic potential of LP MSCs. ( a ) LP MSCs were grown in basal growth medium (DMEM-LG containing 10% FBS) in the presence of t-BHQ (10 μ M) for 7 days. The mRNA expression levels of HO-1 , NQO-1 , ( b ) SIRT1 , and p53 were also analyzed by qRT-PCR. * P

    Article Snippet: Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR

    Proposed models of the effects of OTA or t-BHQ on the regulation and maintenance of MSC stemness via SIRT1. Blocking the nuclear import of NRF2 activates p53, which suppresses SIRT1 promoter activity, resulting in a loss of MSC stemness. Conversely, the protein level of p53 can be decreased by the phosphorylation and nuclear import of NRF2, resulting in the activation of SIRT1 transcription as well as the enhancement of MSC stemness

    Journal: Cell Death & Disease

    Article Title: Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    doi: 10.1038/cddis.2016.3

    Figure Lengend Snippet: Proposed models of the effects of OTA or t-BHQ on the regulation and maintenance of MSC stemness via SIRT1. Blocking the nuclear import of NRF2 activates p53, which suppresses SIRT1 promoter activity, resulting in a loss of MSC stemness. Conversely, the protein level of p53 can be decreased by the phosphorylation and nuclear import of NRF2, resulting in the activation of SIRT1 transcription as well as the enhancement of MSC stemness

    Article Snippet: Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology).

    Techniques: Blocking Assay, Activity Assay, Activation Assay

    t-BHQ induces nuclear import of NRF2 and enhances the self-renewal capacity, but does not affect the osteogenic differentiation of EP MSCs. ( a ) EP MSCs were incubated in basal growth medium (DMEM-LG containing 10% FBS) in the presence of DMSO or t-BHQ (10 μ M) for 16 h. Cell lysates were prepared for the DMSO-treated or t-BHQ-treated EP MSCs, and then fractionated into nuclear and cytosolic extracts. The protein levels of NRF2 and phosphorylated NRF2 were analyzed by western blot analysis for the DMSO- or t-BHQ-treated EP MSCs. The LDH protein level was used as a loading control for cytosolic extracts, and the protein level of LAMIN-B was used as a loading control for nuclear extracts. The expression level of β -CATENIN was also used as a loading control for both cytosolic and nuclear extracts. DMSO was used as a vehicle of t-BHQ. ( b ) Immunofluorescence was performed to observe the nuclear and cytosolic localization of the NRF2 protein in the DMSO- or t-BHQ (10 μ M)-treated EP MSCs. The nuclei were stained with DAPI, and NRF2 was stained with Alexa Fluor 568 (Yellow)-conjugated secondary antibody. The images were obtained using confocal microscopy. Scale bar=100 μ m. ( c ) The mRNA expression levels of HO-1 and NQO-1 were also analyzed with qRT-PCR. * P

    Journal: Cell Death & Disease

    Article Title: Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    doi: 10.1038/cddis.2016.3

    Figure Lengend Snippet: t-BHQ induces nuclear import of NRF2 and enhances the self-renewal capacity, but does not affect the osteogenic differentiation of EP MSCs. ( a ) EP MSCs were incubated in basal growth medium (DMEM-LG containing 10% FBS) in the presence of DMSO or t-BHQ (10 μ M) for 16 h. Cell lysates were prepared for the DMSO-treated or t-BHQ-treated EP MSCs, and then fractionated into nuclear and cytosolic extracts. The protein levels of NRF2 and phosphorylated NRF2 were analyzed by western blot analysis for the DMSO- or t-BHQ-treated EP MSCs. The LDH protein level was used as a loading control for cytosolic extracts, and the protein level of LAMIN-B was used as a loading control for nuclear extracts. The expression level of β -CATENIN was also used as a loading control for both cytosolic and nuclear extracts. DMSO was used as a vehicle of t-BHQ. ( b ) Immunofluorescence was performed to observe the nuclear and cytosolic localization of the NRF2 protein in the DMSO- or t-BHQ (10 μ M)-treated EP MSCs. The nuclei were stained with DAPI, and NRF2 was stained with Alexa Fluor 568 (Yellow)-conjugated secondary antibody. The images were obtained using confocal microscopy. Scale bar=100 μ m. ( c ) The mRNA expression levels of HO-1 and NQO-1 were also analyzed with qRT-PCR. * P

    Article Snippet: Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology).

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

    The phosphorylated level and nuclear localization of NRF2 decrease during prolonged cell passages and osteogenic differentiation. ( a ) EP, LP, undifferentiated, or differentiated MSCs to the osteogenic lineage were harvested at each stage to prepare cell lysates. The mRNA expression of NRF2 was analyzed by qRT-PCR, and ( b ) the protein levels of NRF2 and phosphorylated NRF2 were analyzed by western blot analysis. The expression level of β -CATENIN was used as a loading control. For the same cDNA, the mRNA expression levels of HO-1 ( c ) and NQO-1 ( d ), which are downstream targets of NRF2, were also analyzed by qRT-PCR. * P

    Journal: Cell Death & Disease

    Article Title: Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    doi: 10.1038/cddis.2016.3

    Figure Lengend Snippet: The phosphorylated level and nuclear localization of NRF2 decrease during prolonged cell passages and osteogenic differentiation. ( a ) EP, LP, undifferentiated, or differentiated MSCs to the osteogenic lineage were harvested at each stage to prepare cell lysates. The mRNA expression of NRF2 was analyzed by qRT-PCR, and ( b ) the protein levels of NRF2 and phosphorylated NRF2 were analyzed by western blot analysis. The expression level of β -CATENIN was used as a loading control. For the same cDNA, the mRNA expression levels of HO-1 ( c ) and NQO-1 ( d ), which are downstream targets of NRF2, were also analyzed by qRT-PCR. * P

    Article Snippet: Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Effect of FA-97 on Nrf2/HO-1 signaling in vivo . (A) Representative images of immunohistochemical staining for HO-1 and Nrf2. (B) The expressions of HO-1, NQO-1 and Nrf2 in nuclear were determined by Western Blot. (C) Image pro plus software was used to quantify the IHC images and 10 fields were counted for each mouse. The integrated option density (IOD) of HO-1 and Nrf2 positive cells were shown. (D,E) Densitometric analysis was performed to determine the relative ratios of each protein. β-actin and Lamin A were used as nuclear and cytoplasmic markers, respectively. (F) The expressions of p65 and c-Jun in colonic tissues were detected by IHC and the positive cells were brown. (G) The expression of p65 and c-Jun were determined by Western Blot analysis. (H) Quantification of IHC images by image pro plus software and 10 fields were counted for each mouse. IOD of p65 and c-Jun positive cells were shown. (I) Densitometric analysis was performed to determine the relative ratios of nuclear proteins. Lamin A was used as nuclear marker. (J) Densitometric analysis was performed to determine the relative ratios of p65 and c-Jun in cytoplasm. β-actin was used as cytoplasmic marker. Each experiment was performed at least three times. Scale bars, 200 μm. Data were presented as means ± SD. ## P

    Journal: Frontiers in Immunology

    Article Title: FA-97, a New Synthetic Caffeic Acid Phenethyl Ester Derivative, Ameliorates DSS-Induced Colitis Against Oxidative Stress by Activating Nrf2/HO-1 Pathway

    doi: 10.3389/fimmu.2019.02969

    Figure Lengend Snippet: Effect of FA-97 on Nrf2/HO-1 signaling in vivo . (A) Representative images of immunohistochemical staining for HO-1 and Nrf2. (B) The expressions of HO-1, NQO-1 and Nrf2 in nuclear were determined by Western Blot. (C) Image pro plus software was used to quantify the IHC images and 10 fields were counted for each mouse. The integrated option density (IOD) of HO-1 and Nrf2 positive cells were shown. (D,E) Densitometric analysis was performed to determine the relative ratios of each protein. β-actin and Lamin A were used as nuclear and cytoplasmic markers, respectively. (F) The expressions of p65 and c-Jun in colonic tissues were detected by IHC and the positive cells were brown. (G) The expression of p65 and c-Jun were determined by Western Blot analysis. (H) Quantification of IHC images by image pro plus software and 10 fields were counted for each mouse. IOD of p65 and c-Jun positive cells were shown. (I) Densitometric analysis was performed to determine the relative ratios of nuclear proteins. Lamin A was used as nuclear marker. (J) Densitometric analysis was performed to determine the relative ratios of p65 and c-Jun in cytoplasm. β-actin was used as cytoplasmic marker. Each experiment was performed at least three times. Scale bars, 200 μm. Data were presented as means ± SD. ## P

    Article Snippet: After being incubated in 3% hydrogen peroxide for 10 min, sections were blocked with 100 μl blocking solution for 1 h at room temperature and stained with the following primary antibodies: rabbit monoclonal anti-IL-1β and anti-p65 antibodies (1:100, Cell Signaling Technology, Danvers, MA, USA); rabbit monoclonal anti-IL-6, anti-TNF-α, anti-c-Jun, anti-HO-1, and anti-Nrf2 antibodies (1:200, Abcam, Cambridge, United Kingdom).

    Techniques: In Vivo, Immunohistochemistry, Staining, Western Blot, Software, Expressing, Marker

    Effect of FA-97 on Nrf2/HO-1 signaling pathway in vitro . RAW264.7 cells were pretreated with FA-97 (0, 0.25, 0.5, and 1 μM) for 24 h followed by LPS (1 μg/ml) stimulation for 2 h. (A) The expression of Nrf2 in cytosolic and nuclear extracts were determined by Western Blot. Lamin A and GAPDH were used as nuclear and cytoplasmic markers, respectively. (B) Densitometric analysis was performed to determine the relative ratios of Nrf2. GAPDH and Lamin A were used as nuclear and cytoplasmic markers, respectively. (C) RAW 264.7 cell slides were immune-stained with anti-Nrf2 (green) and DAPI (blue), and then the nuclear translocation of Nrf2 was observed by confocal laser-scanning microscope. (D) After transfected with ARE luciferase reporter plasmid, the Nrf2 transcription activity of RAW 264.7 cells was detected by luciferase activity assay. (E) The level of HO-1, NQO-1, and β-actin were detected by Western Blot. (F) Densitometric analysis was performed to determine the relative ratios of HO-1 and NQO-1. (G) Protein level of p-p65, p65, p-IκBα, IκBα, and β-actin in RAW 264.7 cells were detected by Western Blot. (H) The protein level of p-c-Jun, c-Jun, p-c-Fos, c-Fos, and β-actin in RAW 264.7 cells were detected by Western Blot. (I) Densitometric analysis was performed to determine the relative ratios of each protein. (J) The expression of p65 and c-Jun in cytosolic and nuclear extracts of RAW 264.7 cells were determined by Western Blot. Lamin A and GAPDH were used as nuclear and cytoplasmic markers, respectively. (K) Densitometric analysis was performed to determine the relative ratios of p65 and c-Jun. GAPDH and Lamin A were used as nuclear and cytoplasmic markers, respectively. Scale bars, 20 μm. The results are representative of three independent experiments. # P

    Journal: Frontiers in Immunology

    Article Title: FA-97, a New Synthetic Caffeic Acid Phenethyl Ester Derivative, Ameliorates DSS-Induced Colitis Against Oxidative Stress by Activating Nrf2/HO-1 Pathway

    doi: 10.3389/fimmu.2019.02969

    Figure Lengend Snippet: Effect of FA-97 on Nrf2/HO-1 signaling pathway in vitro . RAW264.7 cells were pretreated with FA-97 (0, 0.25, 0.5, and 1 μM) for 24 h followed by LPS (1 μg/ml) stimulation for 2 h. (A) The expression of Nrf2 in cytosolic and nuclear extracts were determined by Western Blot. Lamin A and GAPDH were used as nuclear and cytoplasmic markers, respectively. (B) Densitometric analysis was performed to determine the relative ratios of Nrf2. GAPDH and Lamin A were used as nuclear and cytoplasmic markers, respectively. (C) RAW 264.7 cell slides were immune-stained with anti-Nrf2 (green) and DAPI (blue), and then the nuclear translocation of Nrf2 was observed by confocal laser-scanning microscope. (D) After transfected with ARE luciferase reporter plasmid, the Nrf2 transcription activity of RAW 264.7 cells was detected by luciferase activity assay. (E) The level of HO-1, NQO-1, and β-actin were detected by Western Blot. (F) Densitometric analysis was performed to determine the relative ratios of HO-1 and NQO-1. (G) Protein level of p-p65, p65, p-IκBα, IκBα, and β-actin in RAW 264.7 cells were detected by Western Blot. (H) The protein level of p-c-Jun, c-Jun, p-c-Fos, c-Fos, and β-actin in RAW 264.7 cells were detected by Western Blot. (I) Densitometric analysis was performed to determine the relative ratios of each protein. (J) The expression of p65 and c-Jun in cytosolic and nuclear extracts of RAW 264.7 cells were determined by Western Blot. Lamin A and GAPDH were used as nuclear and cytoplasmic markers, respectively. (K) Densitometric analysis was performed to determine the relative ratios of p65 and c-Jun. GAPDH and Lamin A were used as nuclear and cytoplasmic markers, respectively. Scale bars, 20 μm. The results are representative of three independent experiments. # P

    Article Snippet: After being incubated in 3% hydrogen peroxide for 10 min, sections were blocked with 100 μl blocking solution for 1 h at room temperature and stained with the following primary antibodies: rabbit monoclonal anti-IL-1β and anti-p65 antibodies (1:100, Cell Signaling Technology, Danvers, MA, USA); rabbit monoclonal anti-IL-6, anti-TNF-α, anti-c-Jun, anti-HO-1, and anti-Nrf2 antibodies (1:200, Abcam, Cambridge, United Kingdom).

    Techniques: In Vitro, Expressing, Western Blot, Staining, Translocation Assay, Laser-Scanning Microscopy, Transfection, Luciferase, Plasmid Preparation, Activity Assay

    The Nrf2/HO-1 pathway was involved in the anti-oxidative and anti-inflammation effects of FA-97 in LPS-induced RAW 264.7 cells. After Nrf2 siRNA transfection, RAW 264.7 cells were treated with FA-97 (0, 0.25, 0.5, and 1 μM) for 24 h followed by LPS (1 μg/ml) stimulation for 2 h. (A) The expression of Nrf2 was detected by Western Blot after Nrf2 siRNA transfection. (B) The level of HO-1, NQO-1, and β-actin were detected by Western Blot. (C) The expression of p65 and c-Jun in nuclear extract were determined and Lamin A was used as nuclear marker. (D) The level of ROS was measured by spectrofluorimeter. The MDA level (E) and the total antioxidant capacity (F) in RAW 264.7 cells were measured according to the kit manufacturer's instructions. (G–I) The concentrations of IL-1β, IL-6, and TNF-α in RAW 264.7 cell culture supernatants were measured by ELISA. (J–M) RAW 264.7 cells were treated with FA-97 (1 μM) for 24 h followed by LPS (1 μg/ml) with or without SnPP (20 μM) stimulation for 2 h. The expression of HO-1 in cells was detected by Western Blot (J) , the expression of p65 and c-Jun in nuclear extract were determined and Lamin A was used as nuclear marker (K) . The total antioxidant capacity in cells were measured according to the kit manufacturer's instructions (L) , as well as the concentration of IL-1β and TNF-α in RAW 264.7 cell culture supernatants were measured by ELISA (M) . The results are representative of three independent experiments and expressed as means ± SD. ## P

    Journal: Frontiers in Immunology

    Article Title: FA-97, a New Synthetic Caffeic Acid Phenethyl Ester Derivative, Ameliorates DSS-Induced Colitis Against Oxidative Stress by Activating Nrf2/HO-1 Pathway

    doi: 10.3389/fimmu.2019.02969

    Figure Lengend Snippet: The Nrf2/HO-1 pathway was involved in the anti-oxidative and anti-inflammation effects of FA-97 in LPS-induced RAW 264.7 cells. After Nrf2 siRNA transfection, RAW 264.7 cells were treated with FA-97 (0, 0.25, 0.5, and 1 μM) for 24 h followed by LPS (1 μg/ml) stimulation for 2 h. (A) The expression of Nrf2 was detected by Western Blot after Nrf2 siRNA transfection. (B) The level of HO-1, NQO-1, and β-actin were detected by Western Blot. (C) The expression of p65 and c-Jun in nuclear extract were determined and Lamin A was used as nuclear marker. (D) The level of ROS was measured by spectrofluorimeter. The MDA level (E) and the total antioxidant capacity (F) in RAW 264.7 cells were measured according to the kit manufacturer's instructions. (G–I) The concentrations of IL-1β, IL-6, and TNF-α in RAW 264.7 cell culture supernatants were measured by ELISA. (J–M) RAW 264.7 cells were treated with FA-97 (1 μM) for 24 h followed by LPS (1 μg/ml) with or without SnPP (20 μM) stimulation for 2 h. The expression of HO-1 in cells was detected by Western Blot (J) , the expression of p65 and c-Jun in nuclear extract were determined and Lamin A was used as nuclear marker (K) . The total antioxidant capacity in cells were measured according to the kit manufacturer's instructions (L) , as well as the concentration of IL-1β and TNF-α in RAW 264.7 cell culture supernatants were measured by ELISA (M) . The results are representative of three independent experiments and expressed as means ± SD. ## P

    Article Snippet: After being incubated in 3% hydrogen peroxide for 10 min, sections were blocked with 100 μl blocking solution for 1 h at room temperature and stained with the following primary antibodies: rabbit monoclonal anti-IL-1β and anti-p65 antibodies (1:100, Cell Signaling Technology, Danvers, MA, USA); rabbit monoclonal anti-IL-6, anti-TNF-α, anti-c-Jun, anti-HO-1, and anti-Nrf2 antibodies (1:200, Abcam, Cambridge, United Kingdom).

    Techniques: Transfection, Expressing, Western Blot, Marker, Multiple Displacement Amplification, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Nrf2/HO-1 signaling is critical for protection against DSS-induced colitis by FA-97. (A) Body weights of mice in each group ( n = 8) were measured. (B) Disease activity index (DAI) of mice in each group was analyzed. (C) Macroscopic appearance of the representative colon from each group. (D) The quantification of colon length from each group of mice. (E) Representative images showing colon pathologic abnormalities with hematoxylin and eosin (H E) staining. Scale bars, 100 μm. (F) The MPO activity in colonic tissues was detected by MPO Activity Assessment Kit using the O-dianisidine method. (G,H) The level of IL-1β, IL-6, and TNF-α in colonic homogenate were measured by ELISA. (I) The total antioxidant capacity in colon were measured according to the kit manufacturer's instructions. (J,K) The expression of HO-1, NQO-1, and Nrf2 in nuclear were determined by Western Blot. (L,M) Densitometric analysis was performed to determine the relative ratios of each protein. β-actin and Lamin A were used as nuclear and cytoplasmic markers, respectively. The results are representative of three independent experiments and expressed as means ± SD. ## P

    Journal: Frontiers in Immunology

    Article Title: FA-97, a New Synthetic Caffeic Acid Phenethyl Ester Derivative, Ameliorates DSS-Induced Colitis Against Oxidative Stress by Activating Nrf2/HO-1 Pathway

    doi: 10.3389/fimmu.2019.02969

    Figure Lengend Snippet: Nrf2/HO-1 signaling is critical for protection against DSS-induced colitis by FA-97. (A) Body weights of mice in each group ( n = 8) were measured. (B) Disease activity index (DAI) of mice in each group was analyzed. (C) Macroscopic appearance of the representative colon from each group. (D) The quantification of colon length from each group of mice. (E) Representative images showing colon pathologic abnormalities with hematoxylin and eosin (H E) staining. Scale bars, 100 μm. (F) The MPO activity in colonic tissues was detected by MPO Activity Assessment Kit using the O-dianisidine method. (G,H) The level of IL-1β, IL-6, and TNF-α in colonic homogenate were measured by ELISA. (I) The total antioxidant capacity in colon were measured according to the kit manufacturer's instructions. (J,K) The expression of HO-1, NQO-1, and Nrf2 in nuclear were determined by Western Blot. (L,M) Densitometric analysis was performed to determine the relative ratios of each protein. β-actin and Lamin A were used as nuclear and cytoplasmic markers, respectively. The results are representative of three independent experiments and expressed as means ± SD. ## P

    Article Snippet: After being incubated in 3% hydrogen peroxide for 10 min, sections were blocked with 100 μl blocking solution for 1 h at room temperature and stained with the following primary antibodies: rabbit monoclonal anti-IL-1β and anti-p65 antibodies (1:100, Cell Signaling Technology, Danvers, MA, USA); rabbit monoclonal anti-IL-6, anti-TNF-α, anti-c-Jun, anti-HO-1, and anti-Nrf2 antibodies (1:200, Abcam, Cambridge, United Kingdom).

    Techniques: Mouse Assay, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

    Proposed mechanistic model of FA-97 ameliorates DSS-induced colitis against oxidative stress by activating Nrf2/HO-1 pathway. Our findings demonstrated a scenario where FA-97 activates Nrf2 and promotes its nuclear translocation, on one hand increasing the expression of its downstream target proteins HO-1 and NQO-1, to reduce the ROS level and enhance the oxidant resistance, on the other hand inhibiting the NF-κB and AP-1 signaling to suppress the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-12), and eventually meliorates inflammatory bowel disease.

    Journal: Frontiers in Immunology

    Article Title: FA-97, a New Synthetic Caffeic Acid Phenethyl Ester Derivative, Ameliorates DSS-Induced Colitis Against Oxidative Stress by Activating Nrf2/HO-1 Pathway

    doi: 10.3389/fimmu.2019.02969

    Figure Lengend Snippet: Proposed mechanistic model of FA-97 ameliorates DSS-induced colitis against oxidative stress by activating Nrf2/HO-1 pathway. Our findings demonstrated a scenario where FA-97 activates Nrf2 and promotes its nuclear translocation, on one hand increasing the expression of its downstream target proteins HO-1 and NQO-1, to reduce the ROS level and enhance the oxidant resistance, on the other hand inhibiting the NF-κB and AP-1 signaling to suppress the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-12), and eventually meliorates inflammatory bowel disease.

    Article Snippet: After being incubated in 3% hydrogen peroxide for 10 min, sections were blocked with 100 μl blocking solution for 1 h at room temperature and stained with the following primary antibodies: rabbit monoclonal anti-IL-1β and anti-p65 antibodies (1:100, Cell Signaling Technology, Danvers, MA, USA); rabbit monoclonal anti-IL-6, anti-TNF-α, anti-c-Jun, anti-HO-1, and anti-Nrf2 antibodies (1:200, Abcam, Cambridge, United Kingdom).

    Techniques: Translocation Assay, Expressing

    Effect of galangin on expression and nuclear translocation of Nrf2. Whole cell lysate and nuclear fractions of HaCaT cells were extracted following treatment with galangin (40 μM) and incubated for 24 h. (A) Western blots were performed using antibody against Nrf2 and phospho-Nrf2. *,# Significantly different from control of Nrf2 and phospho-Nrf2, respectively ( p

    Journal: Biomolecules & Therapeutics

    Article Title: Galangin Activates the ERK/AKT-Driven Nrf2 Signaling Pathway to Increase the Level of Reduced Glutathione in Human Keratinocytes

    doi: 10.4062/biomolther.2016.112

    Figure Lengend Snippet: Effect of galangin on expression and nuclear translocation of Nrf2. Whole cell lysate and nuclear fractions of HaCaT cells were extracted following treatment with galangin (40 μM) and incubated for 24 h. (A) Western blots were performed using antibody against Nrf2 and phospho-Nrf2. *,# Significantly different from control of Nrf2 and phospho-Nrf2, respectively ( p

    Article Snippet: Primary antibodies against TATA-binding protein (TBP) and phospho-Nrf2 were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Translocation Assay, Incubation, Western Blot

    Alpha-lipoic acid (LA) promoted the nuclear translocation of NRF2. The relative nuclear to cytoplasmic NRF2 expression ratio was significantly increased by cisplatin and was further increased by pretreatment with 0.5 mM LA. However, this increase was reversed by siRNA treatment (A), (B). The expression of HO-1 protein in HEI-OC1 cells was also significantly increased by cisplatin and further increased by LA co-treatment, but not increased when cells were treated with siRNA (C), (D). Data are the means ± SD from three independent experiments performed in duplicate. * P

    Journal: PLoS ONE

    Article Title: α-Lipoic acid prevents against cisplatin cytotoxicity via activation of the NRF2/HO-1 antioxidant pathway

    doi: 10.1371/journal.pone.0226769

    Figure Lengend Snippet: Alpha-lipoic acid (LA) promoted the nuclear translocation of NRF2. The relative nuclear to cytoplasmic NRF2 expression ratio was significantly increased by cisplatin and was further increased by pretreatment with 0.5 mM LA. However, this increase was reversed by siRNA treatment (A), (B). The expression of HO-1 protein in HEI-OC1 cells was also significantly increased by cisplatin and further increased by LA co-treatment, but not increased when cells were treated with siRNA (C), (D). Data are the means ± SD from three independent experiments performed in duplicate. * P

    Article Snippet: Previously used NRF2 antibody (abcam) resulted in multi band in western blot and was not good, so we replaced it with a reference in the literature.

    Techniques: Translocation Assay, Expressing

    Effect of NRF2 inhibition on the antioxidant effects of α-lipoic acid (LA) in auditory cells. Reduction of intracellular ROS levels by LA pretreatment was reversed by NRF2 inhibition, demonstrating that LA acts through activation of the NRF2/HO-1 pathway.

    Journal: PLoS ONE

    Article Title: α-Lipoic acid prevents against cisplatin cytotoxicity via activation of the NRF2/HO-1 antioxidant pathway

    doi: 10.1371/journal.pone.0226769

    Figure Lengend Snippet: Effect of NRF2 inhibition on the antioxidant effects of α-lipoic acid (LA) in auditory cells. Reduction of intracellular ROS levels by LA pretreatment was reversed by NRF2 inhibition, demonstrating that LA acts through activation of the NRF2/HO-1 pathway.

    Article Snippet: Previously used NRF2 antibody (abcam) resulted in multi band in western blot and was not good, so we replaced it with a reference in the literature.

    Techniques: Inhibition, Activation Assay

    The effects of α-lipoic acid (LA) on of inflammatory cytokines. With 0.5 mM LA pretreatment, the mRNA levels of proinflammatory cytokines tumor necrosis factor-α (A) and interferon-γ (B) were decreased, and the level of IL-10 (C), an anti-inflammatory cytokine, was increased significantly in HEI-OC1 cells, but this action of LA was offset by NRF2 inhibition. The results of western blots of TNF alpha and IL10 were also similar to the results of PCR (D-F). The data represent the means ± SD of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: α-Lipoic acid prevents against cisplatin cytotoxicity via activation of the NRF2/HO-1 antioxidant pathway

    doi: 10.1371/journal.pone.0226769

    Figure Lengend Snippet: The effects of α-lipoic acid (LA) on of inflammatory cytokines. With 0.5 mM LA pretreatment, the mRNA levels of proinflammatory cytokines tumor necrosis factor-α (A) and interferon-γ (B) were decreased, and the level of IL-10 (C), an anti-inflammatory cytokine, was increased significantly in HEI-OC1 cells, but this action of LA was offset by NRF2 inhibition. The results of western blots of TNF alpha and IL10 were also similar to the results of PCR (D-F). The data represent the means ± SD of three independent experiments. * P

    Article Snippet: Previously used NRF2 antibody (abcam) resulted in multi band in western blot and was not good, so we replaced it with a reference in the literature.

    Techniques: Inhibition, Western Blot, Polymerase Chain Reaction

    SUL-induced activation of Nrf2 in SH-SY5Y cells. Cells were incubated with SUL (5 μ M) for indicated times and nuclear as well as total protein sample extracts were prepared. (a) Nuclear translocation of Nrf2 was monitored by western blotting of nuclear extracts by probing with anti-Nrf2 specific antibody. (b) Nrf2-ARE binding activity was measured by EMSA according to the manufacturer's instruction using biotin-labeled oligonucleotide specific for Nrf2. (c) Phosphorylation of Nrf2 was assessed by western blot analysis with anti-phospho-Nrf2 (Ser40) antibody. The levels of Topo II (a) and actin (c) were examined to ensure equal amount of nuclear and total protein as loading controls, respectively.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Attenuation of β-Amyloid-Induced Oxidative Cell Death by Sulforaphane via Activation of NF-E2-Related Factor 2

    doi: 10.1155/2013/313510

    Figure Lengend Snippet: SUL-induced activation of Nrf2 in SH-SY5Y cells. Cells were incubated with SUL (5 μ M) for indicated times and nuclear as well as total protein sample extracts were prepared. (a) Nuclear translocation of Nrf2 was monitored by western blotting of nuclear extracts by probing with anti-Nrf2 specific antibody. (b) Nrf2-ARE binding activity was measured by EMSA according to the manufacturer's instruction using biotin-labeled oligonucleotide specific for Nrf2. (c) Phosphorylation of Nrf2 was assessed by western blot analysis with anti-phospho-Nrf2 (Ser40) antibody. The levels of Topo II (a) and actin (c) were examined to ensure equal amount of nuclear and total protein as loading controls, respectively.

    Article Snippet: Anti-4-hydroxynonenal (4-HNE) and anti-phospho-Nrf2 antibodies were supplied from Abcam (Cambridge, MA, USA) and Epitomics, Inc. (Burlingame, CA, USA), respectively.

    Techniques: Activation Assay, Incubation, Translocation Assay, Western Blot, Binding Assay, Activity Assay, Labeling

    Schematic diagram that describes neuroprotective effects of SUL against A β -induced oxidative cell death in AD. SUL attenuates A β -induced oxidative damages, pro-apoptotic signals, and apoptotic cell death through the activation of Nrf2-ARE signaling pathway, which consequently fortify Nrf2-dependent antioxidant defense capacity.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Attenuation of β-Amyloid-Induced Oxidative Cell Death by Sulforaphane via Activation of NF-E2-Related Factor 2

    doi: 10.1155/2013/313510

    Figure Lengend Snippet: Schematic diagram that describes neuroprotective effects of SUL against A β -induced oxidative cell death in AD. SUL attenuates A β -induced oxidative damages, pro-apoptotic signals, and apoptotic cell death through the activation of Nrf2-ARE signaling pathway, which consequently fortify Nrf2-dependent antioxidant defense capacity.

    Article Snippet: Anti-4-hydroxynonenal (4-HNE) and anti-phospho-Nrf2 antibodies were supplied from Abcam (Cambridge, MA, USA) and Epitomics, Inc. (Burlingame, CA, USA), respectively.

    Techniques: Activation Assay

    Effect of Nrf2 gene knock-down on SUL-mediated protection against A β 25–35 -induced apoptotic cell death. SH-SY5Y cells were transiently transfected with siRNA of Nrf2 according to the protocol provided by manufacturer and then exposed to A β 25–35 (15 μ M) in the presence or absence of SUL (5 μ M) for 24 h. (a) MTT assay was performed to measure cell viability. Data are represented as mean ± S.D. ( n = 3). ** P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Attenuation of β-Amyloid-Induced Oxidative Cell Death by Sulforaphane via Activation of NF-E2-Related Factor 2

    doi: 10.1155/2013/313510

    Figure Lengend Snippet: Effect of Nrf2 gene knock-down on SUL-mediated protection against A β 25–35 -induced apoptotic cell death. SH-SY5Y cells were transiently transfected with siRNA of Nrf2 according to the protocol provided by manufacturer and then exposed to A β 25–35 (15 μ M) in the presence or absence of SUL (5 μ M) for 24 h. (a) MTT assay was performed to measure cell viability. Data are represented as mean ± S.D. ( n = 3). ** P

    Article Snippet: Anti-4-hydroxynonenal (4-HNE) and anti-phospho-Nrf2 antibodies were supplied from Abcam (Cambridge, MA, USA) and Epitomics, Inc. (Burlingame, CA, USA), respectively.

    Techniques: Transfection, MTT Assay

    Effect of NTG injection on Nrf2 protein levels in the total and nuclear fractions of rat TNC a Representative immunoblots of TNC lysates. Total Nrf2 levels b and nuclear Nrf2 levels c were elevated as early as 0.5 h and persisted for 4 h after NTG injection. β-actin was used as a loading control for total Nrf2. Fibrillarin was used to assess the purity of the nuclear fraction. Data are presented as relative density units normalized to β-actin or Fibrillarin, and expressed as mean ± SD (* P

    Journal: The Journal of Headache and Pain

    Article Title: Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats

    doi: 10.1186/s10194-016-0694-x

    Figure Lengend Snippet: Effect of NTG injection on Nrf2 protein levels in the total and nuclear fractions of rat TNC a Representative immunoblots of TNC lysates. Total Nrf2 levels b and nuclear Nrf2 levels c were elevated as early as 0.5 h and persisted for 4 h after NTG injection. β-actin was used as a loading control for total Nrf2. Fibrillarin was used to assess the purity of the nuclear fraction. Data are presented as relative density units normalized to β-actin or Fibrillarin, and expressed as mean ± SD (* P

    Article Snippet: Sections were incubated with primary antibody against Nrf2 (rabbit polyclonal antibody, dilution 1:200, Abcam, UK), neuronal nuclei NeuN (mouse monoclonal antibody, dilution 1:500, Millipore, USA), c-Fos (rabbit monoclonal antibody, dilution 1:200, Abcam, UK), and nNOS (rabbit monoclonal antibody, dilution 1:200, CST, USA).

    Techniques: Injection, Western Blot

    Co-localization images of Nrf2 with NeuN in TNC. Red indicates Nrf2 immunoreactivity, green indicates NeuN immunoreactivity, and yellow indicates merged signal. In control group a - c of the TNC, Nrf2 is present mainly in the cytoplasm (shown by arrows). In the NTG 2 h group d - f and NTG 4 h group g - i , Nrf2 staining was observed both in the cytoplasm and in the nucleus (shown by arrows). Bar = 50 μm. The arrows indicate cells shown in the top right corner of images c , f , i at about 10 times magnification

    Journal: The Journal of Headache and Pain

    Article Title: Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats

    doi: 10.1186/s10194-016-0694-x

    Figure Lengend Snippet: Co-localization images of Nrf2 with NeuN in TNC. Red indicates Nrf2 immunoreactivity, green indicates NeuN immunoreactivity, and yellow indicates merged signal. In control group a - c of the TNC, Nrf2 is present mainly in the cytoplasm (shown by arrows). In the NTG 2 h group d - f and NTG 4 h group g - i , Nrf2 staining was observed both in the cytoplasm and in the nucleus (shown by arrows). Bar = 50 μm. The arrows indicate cells shown in the top right corner of images c , f , i at about 10 times magnification

    Article Snippet: Sections were incubated with primary antibody against Nrf2 (rabbit polyclonal antibody, dilution 1:200, Abcam, UK), neuronal nuclei NeuN (mouse monoclonal antibody, dilution 1:500, Millipore, USA), c-Fos (rabbit monoclonal antibody, dilution 1:200, Abcam, UK), and nNOS (rabbit monoclonal antibody, dilution 1:200, CST, USA).

    Techniques: Staining

    Effect of sulforaphane (SFN) on the levels of Nrf2 and downstream proteins HO1 and NQO1 in rat TNC 4 h after NTG injection. a Representative immunoblots of TNC. Nuclear Nrf2 b , HO1 c and NQO1 d levels were significantly increased in the SFN plus NTG group compared to those in the H 2 O plus NTG group. Moreover, these protein expressions were increased in the SFN plus control group compared to those in the control group b , c , d . Data are presented as the mean ± SD. (* P

    Journal: The Journal of Headache and Pain

    Article Title: Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats

    doi: 10.1186/s10194-016-0694-x

    Figure Lengend Snippet: Effect of sulforaphane (SFN) on the levels of Nrf2 and downstream proteins HO1 and NQO1 in rat TNC 4 h after NTG injection. a Representative immunoblots of TNC. Nuclear Nrf2 b , HO1 c and NQO1 d levels were significantly increased in the SFN plus NTG group compared to those in the H 2 O plus NTG group. Moreover, these protein expressions were increased in the SFN plus control group compared to those in the control group b , c , d . Data are presented as the mean ± SD. (* P

    Article Snippet: Sections were incubated with primary antibody against Nrf2 (rabbit polyclonal antibody, dilution 1:200, Abcam, UK), neuronal nuclei NeuN (mouse monoclonal antibody, dilution 1:500, Millipore, USA), c-Fos (rabbit monoclonal antibody, dilution 1:200, Abcam, UK), and nNOS (rabbit monoclonal antibody, dilution 1:200, CST, USA).

    Techniques: Injection, Western Blot

    Effect of NTG injection on the levels of Nrf2 downstream proteins HO1 and NQO1 in rat TNC. a Representative immunoblots of TNC. Compared to the NS group or NTG 0 h group, HO1 levels were elevated at 0.5 h, 1 h, 2 h, and 4 h after NTG injection b and NQO1 levels were elevated at 1 h, 2 h, and 4 h after NTG injection c . β-actin was used as the loading control. Data are presented as relative density units normalized to β-actin, and expressed as mean ± SD. (* P

    Journal: The Journal of Headache and Pain

    Article Title: Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats

    doi: 10.1186/s10194-016-0694-x

    Figure Lengend Snippet: Effect of NTG injection on the levels of Nrf2 downstream proteins HO1 and NQO1 in rat TNC. a Representative immunoblots of TNC. Compared to the NS group or NTG 0 h group, HO1 levels were elevated at 0.5 h, 1 h, 2 h, and 4 h after NTG injection b and NQO1 levels were elevated at 1 h, 2 h, and 4 h after NTG injection c . β-actin was used as the loading control. Data are presented as relative density units normalized to β-actin, and expressed as mean ± SD. (* P

    Article Snippet: Sections were incubated with primary antibody against Nrf2 (rabbit polyclonal antibody, dilution 1:200, Abcam, UK), neuronal nuclei NeuN (mouse monoclonal antibody, dilution 1:500, Millipore, USA), c-Fos (rabbit monoclonal antibody, dilution 1:200, Abcam, UK), and nNOS (rabbit monoclonal antibody, dilution 1:200, CST, USA).

    Techniques: Injection, Western Blot

    Decitabine and GO-203 combination decreases expression of DNMT1, 3b, increases Nox4, Duox2, activates Nrf2 and Smad signaling pathway leading to downregulation of c-Myc A–B, H9 and HuT-78 cells were left untreated, treated with 3 uM GO-203 each day for 3 days, single dose of 40 nM Decitabine or the combination. Cells were harvested at 96 hours. Lysates were immunoblotted with the indicated antibodies.

    Journal: Molecular cancer therapeutics

    Article Title: Decitabine Priming Enhances Mucin 1 Inhibition Mediated Disruption of Redox Homeostasis in Cutaneous T-cell Lymphoma

    doi: 10.1158/1535-7163.MCT-17-0060

    Figure Lengend Snippet: Decitabine and GO-203 combination decreases expression of DNMT1, 3b, increases Nox4, Duox2, activates Nrf2 and Smad signaling pathway leading to downregulation of c-Myc A–B, H9 and HuT-78 cells were left untreated, treated with 3 uM GO-203 each day for 3 days, single dose of 40 nM Decitabine or the combination. Cells were harvested at 96 hours. Lysates were immunoblotted with the indicated antibodies.

    Article Snippet: Soluble proteins were analyzed by immunoblotting with anti-DNMT1, 3b (Abcam), anti-Histone 3 (tri methyl K27) (Abcam), anti-TIGAR (Abcam), anti-phospho-p38 (Abcam), anti-Total p38 (Abcam), anti-phospho JNK (Abcam), anti-Total JNK (Abcam), anti-Nrf2 (Abcam), anti-phospho Smad2 and Smad3 (Cell-Signaling Technology), anti-Total Smad2 and Smad3 (Cell-Signaling Technology), anti-c-Myc (Abcam) and anti–GAPDH (Cell-Signaling Technology).

    Techniques: Expressing

    Interleukin-6 activates the Keap1/Nrf2 system in irradiated OSCC cells. ( A ) The images of immunofluorescence of Nrf2 in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. ( B ) The protein levels of Nrf2, phospho-Nrf2, and phospho-STAT3 in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. The whole-cell and nuclear protein were prepared, and the expression of Nrf2, phospho-Nrf2, and phospho-STAT3 proteins was examined using a western blot analysis. ( C ) The expression of Keap1 mRNA in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. ( D ) The protein levels of Keap1 and phospho-p62 in SAS cells. The western blot analysis was done using the same lysate as B . ( E ) The images of immunofluoresent double staining of Keap1 and phospho-p62 in SAS cells at 48 h after 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. The arrows indicate the activated phospho-p62. ( F ) The Nrf2/Keap1 complex formation detected by an immunoprecipitation analysis in SAS cells. After the same treatment as B , the cell lysates were immunoprecipitated using anti-Keap1 antibody. The immunoprecipitates were immunoblotted with anti-Nrf2 antibody.

    Journal: British Journal of Cancer

    Article Title: IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma

    doi: 10.1038/bjc.2016.327

    Figure Lengend Snippet: Interleukin-6 activates the Keap1/Nrf2 system in irradiated OSCC cells. ( A ) The images of immunofluorescence of Nrf2 in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. ( B ) The protein levels of Nrf2, phospho-Nrf2, and phospho-STAT3 in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. The whole-cell and nuclear protein were prepared, and the expression of Nrf2, phospho-Nrf2, and phospho-STAT3 proteins was examined using a western blot analysis. ( C ) The expression of Keap1 mRNA in SAS cells at 48 h after non-X-ray or 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. ( D ) The protein levels of Keap1 and phospho-p62 in SAS cells. The western blot analysis was done using the same lysate as B . ( E ) The images of immunofluoresent double staining of Keap1 and phospho-p62 in SAS cells at 48 h after 10 Gy of X-ray irradiation with or without 200 pg ml −1 IL-6. The arrows indicate the activated phospho-p62. ( F ) The Nrf2/Keap1 complex formation detected by an immunoprecipitation analysis in SAS cells. After the same treatment as B , the cell lysates were immunoprecipitated using anti-Keap1 antibody. The immunoprecipitates were immunoblotted with anti-Nrf2 antibody.

    Article Snippet: Western blot analysis The whole-cell and nuclear protein (Minute Cytoplasmic and Nuclear Extraction Kits; Invent Biotechnologies, Inc., Eden Prairie, MN, USA) were separated using 7.5 or 12.5% SDS–PAGE, transferred onto nitrocellulose membranes, and probed with antibodies against phospho-Nrf2 (S40; 1 : 15 000; Abcam), phospho-STAT3 (Thy705; 1 : 500; Cell Signaling Technology), Keap1 (1 : 1000; Cell Signaling Technology), phospho-p62 (1 : 1000; Medical & Biological Laboratories Co., LTD), Lamin B1 (1 : 10 000; Abcam), and β -actin (1 : 10 000; Sigma, St Louis, MO, USA).

    Techniques: Irradiation, Immunofluorescence, Expressing, Western Blot, Double Staining, Immunoprecipitation

    Interleukin-6 reduces oxidative stress via the Nrf2-antioxidant pathway in irradiated OSCC cells. ( A ) At 48 h after non-irradiation or 10 Gy of X-ray irradiation with vehicle or 200 pg ml −1 IL-6, or 200 pg ml −1 IL-6 plus 20 ng ml −1 tocilizumab, whole-cell lysates in OSCC cells were prepared, and the expression of Mn-SOD and phospho-Nrf2 was examined by a western blot analysis. ( B ) At 48 h after 10 Gy of X-ray irradiation with vehicle or 200 pg ml −1 IL-6, or 200 pg ml −1 IL-6 plus 20 ng ml −1 tocilizumab, the superoxide/ROS were examined in SAS cells using a Cellular ROS/Superoxide Detection Assay Kit. Scale bar, 50 μ m. ( C ) Representative microscopic images of H E and immunohistochemical staining of IL-6, phospho-Nrf2, and Mn-SOD. Scale bar, 25 μ m. ( D ) A schematic illustration of the IL-6-mediated radioresistance model. When OSCC tissue is exposed to X-ray irradiation, the increased level of IL-6 promotes the dissociation of Keap1 and Nrf2 via binding of Keap1 to phospho-p62, leading to Nrf2 activation. At the same time, the IL-6/STAT3 pathway is also activated. The activated Nrf2 and STAT3 then translocate to the nucleus and induce the expression of antioxidant enzymes such as Mn-SOD, and IL-6. The overexpressed IL-6 helps increase the resistance to radiation in both autocrine and paracrine manners in the tumour microenvironment.

    Journal: British Journal of Cancer

    Article Title: IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma

    doi: 10.1038/bjc.2016.327

    Figure Lengend Snippet: Interleukin-6 reduces oxidative stress via the Nrf2-antioxidant pathway in irradiated OSCC cells. ( A ) At 48 h after non-irradiation or 10 Gy of X-ray irradiation with vehicle or 200 pg ml −1 IL-6, or 200 pg ml −1 IL-6 plus 20 ng ml −1 tocilizumab, whole-cell lysates in OSCC cells were prepared, and the expression of Mn-SOD and phospho-Nrf2 was examined by a western blot analysis. ( B ) At 48 h after 10 Gy of X-ray irradiation with vehicle or 200 pg ml −1 IL-6, or 200 pg ml −1 IL-6 plus 20 ng ml −1 tocilizumab, the superoxide/ROS were examined in SAS cells using a Cellular ROS/Superoxide Detection Assay Kit. Scale bar, 50 μ m. ( C ) Representative microscopic images of H E and immunohistochemical staining of IL-6, phospho-Nrf2, and Mn-SOD. Scale bar, 25 μ m. ( D ) A schematic illustration of the IL-6-mediated radioresistance model. When OSCC tissue is exposed to X-ray irradiation, the increased level of IL-6 promotes the dissociation of Keap1 and Nrf2 via binding of Keap1 to phospho-p62, leading to Nrf2 activation. At the same time, the IL-6/STAT3 pathway is also activated. The activated Nrf2 and STAT3 then translocate to the nucleus and induce the expression of antioxidant enzymes such as Mn-SOD, and IL-6. The overexpressed IL-6 helps increase the resistance to radiation in both autocrine and paracrine manners in the tumour microenvironment.

    Article Snippet: Western blot analysis The whole-cell and nuclear protein (Minute Cytoplasmic and Nuclear Extraction Kits; Invent Biotechnologies, Inc., Eden Prairie, MN, USA) were separated using 7.5 or 12.5% SDS–PAGE, transferred onto nitrocellulose membranes, and probed with antibodies against phospho-Nrf2 (S40; 1 : 15 000; Abcam), phospho-STAT3 (Thy705; 1 : 500; Cell Signaling Technology), Keap1 (1 : 1000; Cell Signaling Technology), phospho-p62 (1 : 1000; Medical & Biological Laboratories Co., LTD), Lamin B1 (1 : 10 000; Abcam), and β -actin (1 : 10 000; Sigma, St Louis, MO, USA).

    Techniques: Irradiation, Expressing, Western Blot, Detection Assay, Immunohistochemistry, Staining, Binding Assay, Activation Assay

    ( a ) MC1R-agonist peptide 4 induces serine 40 phosphorylation of Nrf2 after UVA-irradiation ex vivo in a dose-dependent manner. Error bars represent standard error of the mean. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Discovery of a Highly Selective MC1R Agonists Pentapeptide to Be Used as a Skin Pigmentation Enhancer and with Potential Anti-Aging Properties

    doi: 10.3390/ijms20246143

    Figure Lengend Snippet: ( a ) MC1R-agonist peptide 4 induces serine 40 phosphorylation of Nrf2 after UVA-irradiation ex vivo in a dose-dependent manner. Error bars represent standard error of the mean. * p

    Article Snippet: Nrf2 immunostaining was realized on sections from formol-fixed paraffin-embedded skin explants with a monoclonal anti-Nrf2 (phospho S40) antibody (Abcam, # ab76026, clone EP1809Y) diluted at 1:1200 in PBS, BSA 0.3%, Tween20 0.05% for 1 h at room temperature using a Vectastain Kit Vector amplifier system avidin/biotin, and revealed by VIP (Vector laboratories, Ref. SK-4600).

    Techniques: Irradiation, Ex Vivo

    Morphological plasticity is increased in A549 cells stably expressing control or Nrf2 shRNA. A microfabricated μ-Taur device, was used to assay a cell's ability to migrate through narrow 3-diamensional channels of various dimensions 16 hrs after loading. A) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 12 micron posts. Inset shows close ups of posts; B) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 8 micron posts. Inset shows close ups of posts; C D) Migration and plasticity of cells stably expressing Nrf2 shRNA in channels with 12 micron posts (C) or 8 micron posts (D).

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: Morphological plasticity is increased in A549 cells stably expressing control or Nrf2 shRNA. A microfabricated μ-Taur device, was used to assay a cell's ability to migrate through narrow 3-diamensional channels of various dimensions 16 hrs after loading. A) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 12 micron posts. Inset shows close ups of posts; B) Migration and plasticity of cells stably expressing control non-silencing shRNA in channels with 8 micron posts. Inset shows close ups of posts; C D) Migration and plasticity of cells stably expressing Nrf2 shRNA in channels with 12 micron posts (C) or 8 micron posts (D).

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Stable Transfection, Expressing, shRNA, Migration

    Nrf2 is found in a complex with Smad3 and Smad4 and suppresses the activity of a luciferase reporter under control of a synthetic CAGA promoter. A) Immunoprecipitation of endogenous Smad3 from A549 cell lysates followed by immunoblotting for Smad4 and Nrf2. B) FLAG/Smad3 and Nrf2 were either co-transfected or transfected alone into NMuMG cells. FLAG/Smad3 was then immunoprecipitated and immunoblotted for Nrf2. Endogenous Smad3 was immunoprecipitated from H460 cells and then immunoblotted for Nrf2; C) A549 cells were treated with 2 ng/ml TGF beta and solubilized. Cell protein was subjected to GST or GST/Nrf2 pulldown and immunoblotted to pSmad2/3 antibody; D) Wild type A549 cells were transiently co-transfected with a luciferase reporter under control of a synthetic CAGA reporter, a vector expressing β-galactosidase, vectors expressing Smad3, wild type human Nrf2, dominant negative Nrf2, or insertless pcDNA3.1. Reporter activity was determined 72 hrs after transfection.

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: Nrf2 is found in a complex with Smad3 and Smad4 and suppresses the activity of a luciferase reporter under control of a synthetic CAGA promoter. A) Immunoprecipitation of endogenous Smad3 from A549 cell lysates followed by immunoblotting for Smad4 and Nrf2. B) FLAG/Smad3 and Nrf2 were either co-transfected or transfected alone into NMuMG cells. FLAG/Smad3 was then immunoprecipitated and immunoblotted for Nrf2. Endogenous Smad3 was immunoprecipitated from H460 cells and then immunoblotted for Nrf2; C) A549 cells were treated with 2 ng/ml TGF beta and solubilized. Cell protein was subjected to GST or GST/Nrf2 pulldown and immunoblotted to pSmad2/3 antibody; D) Wild type A549 cells were transiently co-transfected with a luciferase reporter under control of a synthetic CAGA reporter, a vector expressing β-galactosidase, vectors expressing Smad3, wild type human Nrf2, dominant negative Nrf2, or insertless pcDNA3.1. Reporter activity was determined 72 hrs after transfection.

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Activity Assay, Luciferase, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation

    Increased R-Smad phosphorylation and CAGA reporter activity in cells expressing Nrf2 shRNA. A) Immunoblot of phospho R-Smads in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; B) Transient co-transfection of a luciferase reporter under control of a synthetic CAGA promoter and a reporter expressing Renilla luciferase into control and Nrf2 shRNA containing A549 cells expressed from retrovirus. Luciferase activity was measured 72 hrs after transfection; C) Immunoblot of PAI-1 expression in control and Nrf2 shRNA containing A549 cells expressed from retrovirus.

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: Increased R-Smad phosphorylation and CAGA reporter activity in cells expressing Nrf2 shRNA. A) Immunoblot of phospho R-Smads in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; B) Transient co-transfection of a luciferase reporter under control of a synthetic CAGA promoter and a reporter expressing Renilla luciferase into control and Nrf2 shRNA containing A549 cells expressed from retrovirus. Luciferase activity was measured 72 hrs after transfection; C) Immunoblot of PAI-1 expression in control and Nrf2 shRNA containing A549 cells expressed from retrovirus.

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Activity Assay, Expressing, shRNA, Cotransfection, Luciferase, Transfection

    RNAi suppression of Nrf2 increases soft agar colony forming ability and cell plasticity. A) Immunoblot of A549 cells stably expressing Nrf2 shRNA from a retrovirus vector (upper) or Nrf2 siRNA from a pSilencer vector (lower), or appropriate control shRNA/siRNA; B) A549 cells stably expressing Nrf2 shRNA from a retrovirus vector or Nrf2 siRNA from a pSilencer vector, or appropriate control shRNA/siRNA were inoculated into soft agar. Five days after inoculation, the numbers of single cells and multi-cellular colonies were quantitated by microscopy; C) Light microscopic images (400 × magnification) of A549 cells stably expressing Nrf2 shRNA or control non-silencing shRNA from a retrovirus vectors.

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: RNAi suppression of Nrf2 increases soft agar colony forming ability and cell plasticity. A) Immunoblot of A549 cells stably expressing Nrf2 shRNA from a retrovirus vector (upper) or Nrf2 siRNA from a pSilencer vector (lower), or appropriate control shRNA/siRNA; B) A549 cells stably expressing Nrf2 shRNA from a retrovirus vector or Nrf2 siRNA from a pSilencer vector, or appropriate control shRNA/siRNA were inoculated into soft agar. Five days after inoculation, the numbers of single cells and multi-cellular colonies were quantitated by microscopy; C) Light microscopic images (400 × magnification) of A549 cells stably expressing Nrf2 shRNA or control non-silencing shRNA from a retrovirus vectors.

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Stable Transfection, Expressing, shRNA, Plasmid Preparation, Microscopy

    Loss of Nrf2 is accompanied by increased motility. A) B) Transwell Migration Assay. The histogram illustrate the number of cells that penetrated the membrane 18 hrs after inoculation; C) D) A scrape assay was used to assess migration. A) C) A549 cells expressing Control or Nrf2 shRNA; B) D) HepG2 cells stably expressing FLAG/luciferase or FLAG/Keap1.

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: Loss of Nrf2 is accompanied by increased motility. A) B) Transwell Migration Assay. The histogram illustrate the number of cells that penetrated the membrane 18 hrs after inoculation; C) D) A scrape assay was used to assess migration. A) C) A549 cells expressing Control or Nrf2 shRNA; B) D) HepG2 cells stably expressing FLAG/luciferase or FLAG/Keap1.

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Transwell Migration Assay, Migration, Expressing, shRNA, Stable Transfection, Luciferase

    Inhibition of Smad3 activity attenuates migration of A549 cells stably expressing Nrf2 shRNA. A) Nrf2 expressing shRNA cells were exposed to 10μM SIS3 for the indicated times. Cells were immunoprecipitated with antibody to pSmad2/3 and then immunoblotted with antibody to pSmad2/3; B-F Cells were subjected to the scrape assay. The assay was performed in the absence (Panel B, C, E) or presence of 10 μM SIS3 (Panels D F). Migration was assessed 24 or 48 hrs after scraping.

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: Inhibition of Smad3 activity attenuates migration of A549 cells stably expressing Nrf2 shRNA. A) Nrf2 expressing shRNA cells were exposed to 10μM SIS3 for the indicated times. Cells were immunoprecipitated with antibody to pSmad2/3 and then immunoblotted with antibody to pSmad2/3; B-F Cells were subjected to the scrape assay. The assay was performed in the absence (Panel B, C, E) or presence of 10 μM SIS3 (Panels D F). Migration was assessed 24 or 48 hrs after scraping.

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Inhibition, Activity Assay, Migration, Stable Transfection, Expressing, shRNA, Immunoprecipitation

    Loss of E-Cadherin is accompanied by increased expression of Slug. A) E-Cadherin immunofluorescence in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; B) Immunoblotting for E-Cadherin in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; C) Immunoblot of Slug expression in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; D) Wild type A549 cells were transiently co-transfected with a luciferase reporter under the control of the human E-Cadherin promoter, a plasmid vector that expressed β-galactosidase, a vector that expressed human wild type Nrf2, or insertless pcDNA3.1. Luciferase/β-Gal activity was measured 72 hrs after transfection.

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: Loss of E-Cadherin is accompanied by increased expression of Slug. A) E-Cadherin immunofluorescence in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; B) Immunoblotting for E-Cadherin in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; C) Immunoblot of Slug expression in control and Nrf2 shRNA containing A549 cells expressed from retrovirus; D) Wild type A549 cells were transiently co-transfected with a luciferase reporter under the control of the human E-Cadherin promoter, a plasmid vector that expressed β-galactosidase, a vector that expressed human wild type Nrf2, or insertless pcDNA3.1. Luciferase/β-Gal activity was measured 72 hrs after transfection.

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Expressing, Immunofluorescence, shRNA, Transfection, Luciferase, Plasmid Preparation, Activity Assay

    Overexpression of Keap1 in HepG2 cells drives down Nrf2 levels and is accompanied by increased colony forming ability, as well as morphological plasticity. A) Immunoblot of cells stably expressing FLAG/Luciferase or FLAG/Keap1; B) Colony forming ability; C) Cell proliferation. Cells expressing FLAG/Luciferase exhibited a doubling time of approximately 31 hrs. Cells expressing FLAG/Keap1 exhibited a doubling time of approximately 24 hrs; D) Light microscopic images (200 × magnification).

    Journal: Oncogene

    Article Title: Increased Cell Migration and Plasticity in Nrf2 Deficient Cancer Cell Lines

    doi: 10.1038/onc.2010.118

    Figure Lengend Snippet: Overexpression of Keap1 in HepG2 cells drives down Nrf2 levels and is accompanied by increased colony forming ability, as well as morphological plasticity. A) Immunoblot of cells stably expressing FLAG/Luciferase or FLAG/Keap1; B) Colony forming ability; C) Cell proliferation. Cells expressing FLAG/Luciferase exhibited a doubling time of approximately 31 hrs. Cells expressing FLAG/Keap1 exhibited a doubling time of approximately 24 hrs; D) Light microscopic images (200 × magnification).

    Article Snippet: Antibodies were obtained as follows: Nrf2 (Santa Cruz: Cat# sc-13032; Abcam: Cat #ab62352), GCLC (Neomarkers: Cat # P1697), β-actin (Sigma: Cat #A5441).

    Techniques: Over Expression, Stable Transfection, Expressing, Luciferase

    Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the prefrontal cortex from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein expression in the prefrontal cortex from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P

    Journal: Frontiers in Pharmacology

    Article Title: Sulforaphane Inhibited the Nociceptive Responses, Anxiety- and Depressive-Like Behaviors Associated With Neuropathic Pain and Improved the Anti-allodynic Effects of Morphine in Mice

    doi: 10.3389/fphar.2018.01332

    Figure Lengend Snippet: Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the prefrontal cortex from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein expression in the prefrontal cortex from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P

    Article Snippet: Proteins were electrophoretically transferred onto PVDF membrane for 120 min, blocked with PBS or TBST + 5% non-fat dry milk or BSA and successively incubated overnight at 4°C with rabbit anti Nrf2 (1:160, Abcam, Cambridge, United Kingdom), HO-1 (1:300, Abcam, Cambridge, United Kingdom), NQO1 (1:333, Sigma, St. Louis, MO, United States), CD11b/c (1:200, Novus Biologicals, Littleton, CO, United States), phospho JNK, total JNK, phospho ERK1/2, total ERK1/2, phospho P38 and total P38 (1:250, Cell Signaling Technology, Danvers, MA, United States), MOR (1:333, Merck, Billerica, MA, United States) or GAPDH antibody (1:5000, Merck, Billerica, MA, United States) which was used as a loading control.

    Techniques: Expressing, Mouse Assay, Western Blot

    Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the spinal cord from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein levels in the ipsilateral site of the spinal cord from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P

    Journal: Frontiers in Pharmacology

    Article Title: Sulforaphane Inhibited the Nociceptive Responses, Anxiety- and Depressive-Like Behaviors Associated With Neuropathic Pain and Improved the Anti-allodynic Effects of Morphine in Mice

    doi: 10.3389/fphar.2018.01332

    Figure Lengend Snippet: Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the spinal cord from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein levels in the ipsilateral site of the spinal cord from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P

    Article Snippet: Proteins were electrophoretically transferred onto PVDF membrane for 120 min, blocked with PBS or TBST + 5% non-fat dry milk or BSA and successively incubated overnight at 4°C with rabbit anti Nrf2 (1:160, Abcam, Cambridge, United Kingdom), HO-1 (1:300, Abcam, Cambridge, United Kingdom), NQO1 (1:333, Sigma, St. Louis, MO, United States), CD11b/c (1:200, Novus Biologicals, Littleton, CO, United States), phospho JNK, total JNK, phospho ERK1/2, total ERK1/2, phospho P38 and total P38 (1:250, Cell Signaling Technology, Danvers, MA, United States), MOR (1:333, Merck, Billerica, MA, United States) or GAPDH antibody (1:5000, Merck, Billerica, MA, United States) which was used as a loading control.

    Techniques: Expressing, Mouse Assay, Western Blot

    Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the hippocampus from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein expression in the hippocampus from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P

    Journal: Frontiers in Pharmacology

    Article Title: Sulforaphane Inhibited the Nociceptive Responses, Anxiety- and Depressive-Like Behaviors Associated With Neuropathic Pain and Improved the Anti-allodynic Effects of Morphine in Mice

    doi: 10.3389/fphar.2018.01332

    Figure Lengend Snippet: Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the hippocampus from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A) , HO-1 (B) , NQO1 (C) , CD11b/c (D) , and MOR (E) protein expression in the hippocampus from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F) . In all panels, ∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice ( P

    Article Snippet: Proteins were electrophoretically transferred onto PVDF membrane for 120 min, blocked with PBS or TBST + 5% non-fat dry milk or BSA and successively incubated overnight at 4°C with rabbit anti Nrf2 (1:160, Abcam, Cambridge, United Kingdom), HO-1 (1:300, Abcam, Cambridge, United Kingdom), NQO1 (1:333, Sigma, St. Louis, MO, United States), CD11b/c (1:200, Novus Biologicals, Littleton, CO, United States), phospho JNK, total JNK, phospho ERK1/2, total ERK1/2, phospho P38 and total P38 (1:250, Cell Signaling Technology, Danvers, MA, United States), MOR (1:333, Merck, Billerica, MA, United States) or GAPDH antibody (1:5000, Merck, Billerica, MA, United States) which was used as a loading control.

    Techniques: Expressing, Mouse Assay, Western Blot

    The involvement of NRF2 signaling in the downregulation of IL-13- and TGF- β 1-induced periostin expression by cinnamaldehyde. (a, b) Knockdown of NRF2 reverses the effects of cinnamaldehyde (CIN) on TGF- β 1-induced expression of periostin (POSTN) mRNA and protein. (c, d) Knockdown of NRF2 reverses the effects of CIN on IL-13-induced expression of POSTN mRNA and protein. NHDF cells were transiently transfected with control siRNA or NRF2 siRNA for 24 h; next, cells were precultured in 25 μ M CIN for 30 min and then treated with 5 ng/mL TGF- β 1 or 50 ng/mL IL-13 or left untreated for 48 h. Expression levels of POSTN mRNA and protein in the cell supernatants are depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: NRF2 Activation Inhibits Both TGF-β1- and IL-13-Mediated Periostin Expression in Fibroblasts: Benefit of Cinnamaldehyde for Antifibrotic Treatment

    doi: 10.1155/2018/2475047

    Figure Lengend Snippet: The involvement of NRF2 signaling in the downregulation of IL-13- and TGF- β 1-induced periostin expression by cinnamaldehyde. (a, b) Knockdown of NRF2 reverses the effects of cinnamaldehyde (CIN) on TGF- β 1-induced expression of periostin (POSTN) mRNA and protein. (c, d) Knockdown of NRF2 reverses the effects of CIN on IL-13-induced expression of POSTN mRNA and protein. NHDF cells were transiently transfected with control siRNA or NRF2 siRNA for 24 h; next, cells were precultured in 25 μ M CIN for 30 min and then treated with 5 ng/mL TGF- β 1 or 50 ng/mL IL-13 or left untreated for 48 h. Expression levels of POSTN mRNA and protein in the cell supernatants are depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Article Snippet: Samples were incubated with a primary rabbit anti-NRF2 antibody or control IgG (Abcam, Cambridge, UK).

    Techniques: Expressing, Transfection

    Schematic model of the molecular mechanism by which cinnamaldehyde inhibits both IL-13- and TGF- β 1-mediated periostin expression in fibroblasts. Cinnamaldehyde induces NRF2 nuclear translocation and HMOX1 production, which downregulates IL-13- and TGF- β 1-mediated POSTN , TNC , VEGF , and CTGF expression by reducing ROS production, followed by the attenuation of fibrosis.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: NRF2 Activation Inhibits Both TGF-β1- and IL-13-Mediated Periostin Expression in Fibroblasts: Benefit of Cinnamaldehyde for Antifibrotic Treatment

    doi: 10.1155/2018/2475047

    Figure Lengend Snippet: Schematic model of the molecular mechanism by which cinnamaldehyde inhibits both IL-13- and TGF- β 1-mediated periostin expression in fibroblasts. Cinnamaldehyde induces NRF2 nuclear translocation and HMOX1 production, which downregulates IL-13- and TGF- β 1-mediated POSTN , TNC , VEGF , and CTGF expression by reducing ROS production, followed by the attenuation of fibrosis.

    Article Snippet: Samples were incubated with a primary rabbit anti-NRF2 antibody or control IgG (Abcam, Cambridge, UK).

    Techniques: Expressing, Translocation Assay

    Knockdown of NRF2 upregulates the expression of periostin in dermal fibroblasts. (a) NHDF cells were transiently transfected with control siRNA or NRF2 siRNA for 24 h, after which whole cell lysates or total RNA were extracted. The expression levels of NRF2 were normalized to that of GAPDH . NRF2 protein and mRNA expressions are depicted. (b, c) NHDF cells were transiently transfected with control siRNA or 5 nM NRF2 siRNA for 24 h and then stimulated with 5 ng/mL TGF- β 1 (b) or IL-13 (c). POSTN mRNA expression is depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: NRF2 Activation Inhibits Both TGF-β1- and IL-13-Mediated Periostin Expression in Fibroblasts: Benefit of Cinnamaldehyde for Antifibrotic Treatment

    doi: 10.1155/2018/2475047

    Figure Lengend Snippet: Knockdown of NRF2 upregulates the expression of periostin in dermal fibroblasts. (a) NHDF cells were transiently transfected with control siRNA or NRF2 siRNA for 24 h, after which whole cell lysates or total RNA were extracted. The expression levels of NRF2 were normalized to that of GAPDH . NRF2 protein and mRNA expressions are depicted. (b, c) NHDF cells were transiently transfected with control siRNA or 5 nM NRF2 siRNA for 24 h and then stimulated with 5 ng/mL TGF- β 1 (b) or IL-13 (c). POSTN mRNA expression is depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Article Snippet: Samples were incubated with a primary rabbit anti-NRF2 antibody or control IgG (Abcam, Cambridge, UK).

    Techniques: Expressing, Transfection

    The involvement of NRF2 signaling in the downregulation of IL-13- and TGF- β 1-induced tenascin-C, VEGF, and CTGF mRNA expression by cinnamaldehyde. (a–c) Knockdown of NRF2 reverses the effects of cinnamaldehyde (CIN) on TGF- β 1-induced mRNA expression of TNC , VEGF , and CTGF . (d–f) Knockdown of NRF2 reverses the effects of CIN on IL-13-induced mRNA expression of TNC , VEGF , and CTGF . NHDF cells were transiently transfected with control siRNA or 5 nM NRF2 siRNA for 24 h; next, cells were precultured with 25 μ M CIN for 30 min and then treated with 5 ng/mL TGF- β 1 or 50 ng/mL IL-13 or left untreated for 48 h. mRNA expression levels of TNC , VEGF , and CTGF are depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: NRF2 Activation Inhibits Both TGF-β1- and IL-13-Mediated Periostin Expression in Fibroblasts: Benefit of Cinnamaldehyde for Antifibrotic Treatment

    doi: 10.1155/2018/2475047

    Figure Lengend Snippet: The involvement of NRF2 signaling in the downregulation of IL-13- and TGF- β 1-induced tenascin-C, VEGF, and CTGF mRNA expression by cinnamaldehyde. (a–c) Knockdown of NRF2 reverses the effects of cinnamaldehyde (CIN) on TGF- β 1-induced mRNA expression of TNC , VEGF , and CTGF . (d–f) Knockdown of NRF2 reverses the effects of CIN on IL-13-induced mRNA expression of TNC , VEGF , and CTGF . NHDF cells were transiently transfected with control siRNA or 5 nM NRF2 siRNA for 24 h; next, cells were precultured with 25 μ M CIN for 30 min and then treated with 5 ng/mL TGF- β 1 or 50 ng/mL IL-13 or left untreated for 48 h. mRNA expression levels of TNC , VEGF , and CTGF are depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Article Snippet: Samples were incubated with a primary rabbit anti-NRF2 antibody or control IgG (Abcam, Cambridge, UK).

    Techniques: Expressing, Transfection

    Activation of the NRF2/HMOX1 and NQO-1 pathway by cinnamaldehyde. (a) Effects of cinnamaldehyde (CIN) on the viability of NHDFs. (b) NHDF cells were treated with DMSO (control) or CIN (25 μ M) for 3 h and fixed. Cells were then stained with the anti-NRF2 antibody or control IgG (green) and DAPI (blue) and visualized by fluorescence microscopy. (c–e) NHDF cells were treated with DMSO (control) or CIN (25 μ M) for 6 h, after which total RNA was extracted. (c) NRF2 mRNA expression, (d) HMOX1 mRNA expression, and (e) NQO1 mRNA expression are depicted. (f) NHDF cells were treated with 100% ethanol (control) or CIN (25 μ M) for 24 h, after which whole cell lysates were extracted. The expression of HMOX1, NQO1, and GAPDH was depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: NRF2 Activation Inhibits Both TGF-β1- and IL-13-Mediated Periostin Expression in Fibroblasts: Benefit of Cinnamaldehyde for Antifibrotic Treatment

    doi: 10.1155/2018/2475047

    Figure Lengend Snippet: Activation of the NRF2/HMOX1 and NQO-1 pathway by cinnamaldehyde. (a) Effects of cinnamaldehyde (CIN) on the viability of NHDFs. (b) NHDF cells were treated with DMSO (control) or CIN (25 μ M) for 3 h and fixed. Cells were then stained with the anti-NRF2 antibody or control IgG (green) and DAPI (blue) and visualized by fluorescence microscopy. (c–e) NHDF cells were treated with DMSO (control) or CIN (25 μ M) for 6 h, after which total RNA was extracted. (c) NRF2 mRNA expression, (d) HMOX1 mRNA expression, and (e) NQO1 mRNA expression are depicted. (f) NHDF cells were treated with 100% ethanol (control) or CIN (25 μ M) for 24 h, after which whole cell lysates were extracted. The expression of HMOX1, NQO1, and GAPDH was depicted. The values were adjusted by GAPDH expression. The same experiments were performed three times. ∗ P

    Article Snippet: Samples were incubated with a primary rabbit anti-NRF2 antibody or control IgG (Abcam, Cambridge, UK).

    Techniques: Activation Assay, Staining, Fluorescence, Microscopy, Expressing

    A 1 -containing U. lactuca fraction (fraction 3*, see Material and methods) comparably induced the cytoprotective genes through the Nrf2–ARE pathway. (A) Fraction 3* increased endogenous NQO1 mRNA levels in human IMR-32 cells after 12 h of treatment

    Journal: Free radical biology & medicine

    Article Title: Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2-ARE pathway

    doi: 10.1016/j.freeradbiomed.2012.12.019

    Figure Lengend Snippet: A 1 -containing U. lactuca fraction (fraction 3*, see Material and methods) comparably induced the cytoprotective genes through the Nrf2–ARE pathway. (A) Fraction 3* increased endogenous NQO1 mRNA levels in human IMR-32 cells after 12 h of treatment

    Article Snippet: Anti-NQO1 (mouse) and anti-Nrf2 (rabbit) antibodies were from Abcam; anti-hemagglutinin (HA) (mouse) was from Covance; anti- β -actin (rabbit), anti- α -tubulin (rabbit), anti-mouse–HRP, and anti-rabbit-HRP antibodies were from Cell Signaling Technology; anti-Oct-1 (C-21), anti-Gal4 (DBD), and anti-Keap1 (E-20) were from Santa Cruz Biotechnology; and anti-goat-HRP was from Chemicon (Millipore).

    Techniques:

    C18 acid 1 induces cytoprotective genes in IMR-32 cells. (A) Treatment with 1 led to dose-dependent increase in NQO1, but not NRF2, mRNA levels after 12 h ( n =3). (B) At the highest active concentration (10 μ/ml) 1 induced multiple other ARE-Nrf2-regulated

    Journal: Free radical biology & medicine

    Article Title: Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2-ARE pathway

    doi: 10.1016/j.freeradbiomed.2012.12.019

    Figure Lengend Snippet: C18 acid 1 induces cytoprotective genes in IMR-32 cells. (A) Treatment with 1 led to dose-dependent increase in NQO1, but not NRF2, mRNA levels after 12 h ( n =3). (B) At the highest active concentration (10 μ/ml) 1 induced multiple other ARE-Nrf2-regulated

    Article Snippet: Anti-NQO1 (mouse) and anti-Nrf2 (rabbit) antibodies were from Abcam; anti-hemagglutinin (HA) (mouse) was from Covance; anti- β -actin (rabbit), anti- α -tubulin (rabbit), anti-mouse–HRP, and anti-rabbit-HRP antibodies were from Cell Signaling Technology; anti-Oct-1 (C-21), anti-Gal4 (DBD), and anti-Keap1 (E-20) were from Santa Cruz Biotechnology; and anti-goat-HRP was from Chemicon (Millipore).

    Techniques: Concentration Assay

    C18 acid 1 requires Nrf2 and PI3K for the induction of ARE-regulated genes in IMR-32 cells. (A, B) Nrf2 is essential for 1 -induced NQO1 expression at both (A) the transcript and (B) the protein levels. The cells were incubated for 48 h after siRNA transfection

    Journal: Free radical biology & medicine

    Article Title: Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2-ARE pathway

    doi: 10.1016/j.freeradbiomed.2012.12.019

    Figure Lengend Snippet: C18 acid 1 requires Nrf2 and PI3K for the induction of ARE-regulated genes in IMR-32 cells. (A, B) Nrf2 is essential for 1 -induced NQO1 expression at both (A) the transcript and (B) the protein levels. The cells were incubated for 48 h after siRNA transfection

    Article Snippet: Anti-NQO1 (mouse) and anti-Nrf2 (rabbit) antibodies were from Abcam; anti-hemagglutinin (HA) (mouse) was from Covance; anti- β -actin (rabbit), anti- α -tubulin (rabbit), anti-mouse–HRP, and anti-rabbit-HRP antibodies were from Cell Signaling Technology; anti-Oct-1 (C-21), anti-Gal4 (DBD), and anti-Keap1 (E-20) were from Santa Cruz Biotechnology; and anti-goat-HRP was from Chemicon (Millipore).

    Techniques: Expressing, Incubation, Transfection

    Nrf2 expression in both cytoplasm and nucleus in the liver

    Journal: Gastroenterology and hepatology

    Article Title: Salutary effect of pre-treatment with an Nrf2 inducer on ischemia reperfusion injury in the rat liver

    doi: 10.3968/5206

    Figure Lengend Snippet: Nrf2 expression in both cytoplasm and nucleus in the liver

    Article Snippet: Target proteins were measured by western blot analysis with the following antibodies: rabbit antibodies against Nrf2 (1:200 dilution, Abcam), GCLM (1:1000 dilution, abcam), GCLC (1:800 dilution, abcam), and GAPDH (1:5000 dilution, Sigma).

    Techniques: Expressing

    Paeonin exerted an anti-oxidative role by PI3K/Akt-mediated Nrf2 signaling pathway. ( A ) Cell viability was tested by MTT assay in H 2 O 2 -incubated GES-1 cells with 200 μg/ml Paeonin for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. The cell viability presented a time-dependent increase. ( B ) PI3K/Akt-mediated Keap-1 Nrf2 signaling pathway-related protein expressions were measured by WB in H 2 O 2 -exposed GES-1 cells with 200 μg/ml Paeonin for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. ( C ) The location expression of Nrf2 was examined by IF in H 2 O 2 -stimulated GES-1 cells with 200 μg/ml Paeonin for 2 h, 8 h and 12 h. The cell nucleus was stained into blue, while Nrf2 protein was stained into red. ( D ) GSH content was detected in the GSE-1, H 2 O 2 , H 2 O 2 + Paeonin and H 2 O 2 + Paeonin + GSK690693 groups. GSH content in the three experiment groups was higher than that in the GSE-1 group; * P

    Journal: Scientific Reports

    Article Title: Paeonin extracted from potatoes protects gastric epithelial cells from H2O2-induced oxidative damage in vitro by PI3K/Akt-mediated Nrf2 signaling pathway

    doi: 10.1038/s41598-018-28772-5

    Figure Lengend Snippet: Paeonin exerted an anti-oxidative role by PI3K/Akt-mediated Nrf2 signaling pathway. ( A ) Cell viability was tested by MTT assay in H 2 O 2 -incubated GES-1 cells with 200 μg/ml Paeonin for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. The cell viability presented a time-dependent increase. ( B ) PI3K/Akt-mediated Keap-1 Nrf2 signaling pathway-related protein expressions were measured by WB in H 2 O 2 -exposed GES-1 cells with 200 μg/ml Paeonin for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. ( C ) The location expression of Nrf2 was examined by IF in H 2 O 2 -stimulated GES-1 cells with 200 μg/ml Paeonin for 2 h, 8 h and 12 h. The cell nucleus was stained into blue, while Nrf2 protein was stained into red. ( D ) GSH content was detected in the GSE-1, H 2 O 2 , H 2 O 2 + Paeonin and H 2 O 2 + Paeonin + GSK690693 groups. GSH content in the three experiment groups was higher than that in the GSE-1 group; * P

    Article Snippet: After running for 1.5 h at a constant voltage of 120 V, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes for 2 h at a constant current of 200 mA, blocked in 5% non-fat milk for 2 h at room temperature, and incubated with rabbit anti-Nrf2 primary antibody (1:1000 dilution; Abcam, USA), rabbit anti-HO-1 primary antibody (1:1000 dilution; Abcam, USA), rabbit anti-NQO1 primary antibody (1:2000 dilution; Abcam, USA), rabbit anti-phospho-Akt (p-Akt) primary antibody (1:1000 dilution; Abcam, USA), rabbit anti-Akt primary antibody (1:2000 dilution; Abcam, USA), or mouse anti-β-actin (1:1000 dilution, as an internal control; Abcam, USA) overnight at 4 °C.

    Techniques: MTT Assay, Incubation, Western Blot, Expressing, Staining

    Roles of FA and Bz in the exhibition of cell proliferation and the promotion of EMT or in the prevention of apoptosis. ① FA and Bz activate the intracellular ROS of JEG-3, ② which in turn activates oxidative stress. ③ This activates apoptosis by activating apoptosis-related eIF2, or by activating the antioxidant factor, Nrf2, ④ to regulate the activation of ROS by inducing an antioxidant effect through the increase of Nrf2 which blocks the activation of ROS. However, FA and Bz not only activate ROS, but also increase the activity of Nrf2, which prevents the activation of oxidative stress and apoptosis. ⑤ In addition, FA and Bz induce cell proliferation and EMT, either directly or indirectly through Nrf2. Therefore, FA and Bz inhibit apoptosis through the ROS-Nrf2 pathway, leading to an increase in proliferation and EMT.

    Journal: International Journal of Environmental Research and Public Health

    Article Title: Treatment of Human Placental Choriocarcinoma Cells with Formaldehyde and Benzene Induced Growth and Epithelial Mesenchymal Transition via Induction of an Antioxidant Effect

    doi: 10.3390/ijerph14080854

    Figure Lengend Snippet: Roles of FA and Bz in the exhibition of cell proliferation and the promotion of EMT or in the prevention of apoptosis. ① FA and Bz activate the intracellular ROS of JEG-3, ② which in turn activates oxidative stress. ③ This activates apoptosis by activating apoptosis-related eIF2, or by activating the antioxidant factor, Nrf2, ④ to regulate the activation of ROS by inducing an antioxidant effect through the increase of Nrf2 which blocks the activation of ROS. However, FA and Bz not only activate ROS, but also increase the activity of Nrf2, which prevents the activation of oxidative stress and apoptosis. ⑤ In addition, FA and Bz induce cell proliferation and EMT, either directly or indirectly through Nrf2. Therefore, FA and Bz inhibit apoptosis through the ROS-Nrf2 pathway, leading to an increase in proliferation and EMT.

    Article Snippet: The membrane was then incubated overnight at 4 °C with mouse monoclonal anti-GAPDH antibody (Abcam plc, Cambridge, UK), mouse monoclonal anti-cyclin D1 antibody (Abcam plc.), rabbit polyclonal anti-cyclin E1 antibody (Abcam plc.), mouse monoclonal anti-p21 antibody (Abcam plc.), rabbit monoclonal anti-p27 antibody (Abcam plc.), mouse monoclonal anti-N-cadherin antibody (Abcam plc.), mouse monoclonal anti-snail antibody (Cell Signaling Technology, Inc, Danvers, MA, USA), mouse monoclonal anti-slug antibody (Abcam plc.), rabbit monoclonal anti-Nrf2 (Abcam plc.), and rabbit polyclonal anti-phospho-eIF2α antibody (Cell Signaling).

    Techniques: Activation Assay, Activity Assay

    Effect of FA and Bz on protein expression of antioxidant factor nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) and endoplasmic reticulum (ER) stress marker eIF2α in JEG-3. JEG-3 cells were seeded in 100 mm dishes and treated with medium containing 0.1% DMSO (control), ( A ) FA (10 −11 M and 10 −8 M), or ( B ) Bz (10 −8 M and 10 −5 M) for 72 h. After protein extraction, Western blot was conducted to confirm the protein expression of the antioxidant factor, Nrf2, the ER stress marker, eIF2α, and the housekeeping gene, GAPDH. Quantification of Nrf2 and eIF2α protein was conducted by measuring band densities using a CS analyzer 4 (ATTO, Corp., Japan), and their protein levels were then normalized by the band value of GAPDH. Values shown are the means ± SD. * mean values were significantly different from 0.1% DMSO (control), p

    Journal: International Journal of Environmental Research and Public Health

    Article Title: Treatment of Human Placental Choriocarcinoma Cells with Formaldehyde and Benzene Induced Growth and Epithelial Mesenchymal Transition via Induction of an Antioxidant Effect

    doi: 10.3390/ijerph14080854

    Figure Lengend Snippet: Effect of FA and Bz on protein expression of antioxidant factor nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) and endoplasmic reticulum (ER) stress marker eIF2α in JEG-3. JEG-3 cells were seeded in 100 mm dishes and treated with medium containing 0.1% DMSO (control), ( A ) FA (10 −11 M and 10 −8 M), or ( B ) Bz (10 −8 M and 10 −5 M) for 72 h. After protein extraction, Western blot was conducted to confirm the protein expression of the antioxidant factor, Nrf2, the ER stress marker, eIF2α, and the housekeeping gene, GAPDH. Quantification of Nrf2 and eIF2α protein was conducted by measuring band densities using a CS analyzer 4 (ATTO, Corp., Japan), and their protein levels were then normalized by the band value of GAPDH. Values shown are the means ± SD. * mean values were significantly different from 0.1% DMSO (control), p

    Article Snippet: The membrane was then incubated overnight at 4 °C with mouse monoclonal anti-GAPDH antibody (Abcam plc, Cambridge, UK), mouse monoclonal anti-cyclin D1 antibody (Abcam plc.), rabbit polyclonal anti-cyclin E1 antibody (Abcam plc.), mouse monoclonal anti-p21 antibody (Abcam plc.), rabbit monoclonal anti-p27 antibody (Abcam plc.), mouse monoclonal anti-N-cadherin antibody (Abcam plc.), mouse monoclonal anti-snail antibody (Cell Signaling Technology, Inc, Danvers, MA, USA), mouse monoclonal anti-slug antibody (Abcam plc.), rabbit monoclonal anti-Nrf2 (Abcam plc.), and rabbit polyclonal anti-phospho-eIF2α antibody (Cell Signaling).

    Techniques: Expressing, Marker, Protein Extraction, Western Blot

    Iron overload inhibits NF-E2-related factor 2 (Nrf2) expression in PC12 cells after 48 hours of culture. (A) Quantification of Nrf2 mRNA in PC12 cells in the different groups as shown by quantitative PCR. â-Actin mRNA served as an internal standard. The relative levels of Nrf2 mRNA were analyzed using the comparative threshold cycle method (2 -ÄÄCt ). (B) Western blot assay results showing Nrf2 protein expression in PC12 cells. Nrf2 protein level is expressed as the absorbance ratio to â-actin. (C) Immunofluorescence microscopy on PC12 cells showing that Nrf2 localizes both in the cytoplasm and nucleus, and that it is primarily localized in the nucleus in the NGG and HGG. In the 400 μmol/L FAC group, the fluorescence intensity was reduced and labeling was dispersed. Deferoxamine promoted the expression of Nrf2 in the FAC + DFO group. (A, B) Data are shown as mean ± SD from triplicate experiments. One-way analysis of variance was adopted for multiple-group comparison. Two-tailed Student's t -test was used for intergroup comparison. a P

    Journal: Neural Regeneration Research

    Article Title: Neurotoxic effects of iron overload under high glucose concentration

    doi: 10.3969/j.issn.1673-5374.2013.36.008

    Figure Lengend Snippet: Iron overload inhibits NF-E2-related factor 2 (Nrf2) expression in PC12 cells after 48 hours of culture. (A) Quantification of Nrf2 mRNA in PC12 cells in the different groups as shown by quantitative PCR. â-Actin mRNA served as an internal standard. The relative levels of Nrf2 mRNA were analyzed using the comparative threshold cycle method (2 -ÄÄCt ). (B) Western blot assay results showing Nrf2 protein expression in PC12 cells. Nrf2 protein level is expressed as the absorbance ratio to â-actin. (C) Immunofluorescence microscopy on PC12 cells showing that Nrf2 localizes both in the cytoplasm and nucleus, and that it is primarily localized in the nucleus in the NGG and HGG. In the 400 μmol/L FAC group, the fluorescence intensity was reduced and labeling was dispersed. Deferoxamine promoted the expression of Nrf2 in the FAC + DFO group. (A, B) Data are shown as mean ± SD from triplicate experiments. One-way analysis of variance was adopted for multiple-group comparison. Two-tailed Student's t -test was used for intergroup comparison. a P

    Article Snippet: A total of 25 μg protein from each sample was resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA), blocked, incubated in primary rabbit anti-Nrf2 monoclonal antibody (1:400; ab54364, Abcam, Cambridge, UK) and goat anti-β-actin polyclonal antibody (1:500; ab8229, Abcam) overnight at 4°C.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Microscopy, Fluorescence, Labeling, Two Tailed Test

    The expression of Nrf2 in kidney tissues of different groups was detected by immunofluorescence staining under a laser scanning confocal microscope (magnification 400x). (a) Group S; (b) group M; (c) group D1; (d) group D2; (e) group B1; (f) group B2; and (g) group B3. Positive Nrf2 cells were stained green, with the sections counterstained with 4,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Arrows indicate Nrf2 positive glomerular cells, while the triangle points at Nrf2 positive tubular epithelial cells.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Dexmedetomidine Pretreatment Attenuates Kidney Injury and Oxidative Stress during Orthotopic Autologous Liver Transplantation in Rats

    doi: 10.1155/2016/4675817

    Figure Lengend Snippet: The expression of Nrf2 in kidney tissues of different groups was detected by immunofluorescence staining under a laser scanning confocal microscope (magnification 400x). (a) Group S; (b) group M; (c) group D1; (d) group D2; (e) group B1; (f) group B2; and (g) group B3. Positive Nrf2 cells were stained green, with the sections counterstained with 4,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Arrows indicate Nrf2 positive glomerular cells, while the triangle points at Nrf2 positive tubular epithelial cells.

    Article Snippet: Rabbit monoclonal anti-Nrf2 antibody (Abcam, UK) was added to the slices and incubated at 4°C overnight; FITC-labeled goat anti-rabbit IgG antibody was added for 2 h and mounted with antifluorescence quenching coverslip which contained DAPI.

    Techniques: Expressing, Immunofluorescence, Staining, Microscopy

    The proportion of Nrf2 positive cells and semiquantitative immunofluorescence of Nrf2 in glomerular cells or tubular epithelial cells in kidney tissues of different groups. The cells were counted under a laser scanning confocal microscope (magnification 400x). (a) The proportion of Nrf2 positive cells in tubular epithelial cells; (b) the proportion of Nrf2 positive cells in glomerular cells; (c) semiquantitative immunofluorescence of Nrf2 in tubular epithelial cells; (d) semiquantitative immunofluorescence of Nrf2 in glomerular cells. Relative immunofluorescence intensities were normalized to that of group S. The data were represented as mean ± standard deviation (SD), n = 8. ▲ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Dexmedetomidine Pretreatment Attenuates Kidney Injury and Oxidative Stress during Orthotopic Autologous Liver Transplantation in Rats

    doi: 10.1155/2016/4675817

    Figure Lengend Snippet: The proportion of Nrf2 positive cells and semiquantitative immunofluorescence of Nrf2 in glomerular cells or tubular epithelial cells in kidney tissues of different groups. The cells were counted under a laser scanning confocal microscope (magnification 400x). (a) The proportion of Nrf2 positive cells in tubular epithelial cells; (b) the proportion of Nrf2 positive cells in glomerular cells; (c) semiquantitative immunofluorescence of Nrf2 in tubular epithelial cells; (d) semiquantitative immunofluorescence of Nrf2 in glomerular cells. Relative immunofluorescence intensities were normalized to that of group S. The data were represented as mean ± standard deviation (SD), n = 8. ▲ P

    Article Snippet: Rabbit monoclonal anti-Nrf2 antibody (Abcam, UK) was added to the slices and incubated at 4°C overnight; FITC-labeled goat anti-rabbit IgG antibody was added for 2 h and mounted with antifluorescence quenching coverslip which contained DAPI.

    Techniques: Immunofluorescence, Microscopy, Standard Deviation

    Deletion of Nrf2 Leads to Increased Sema6A Expression in Ischemic Inner Retina in OIR.

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Deletion of Nrf2 Leads to Increased Sema6A Expression in Ischemic Inner Retina in OIR.

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Expressing

    Inhibition of Sema6A abrogates the restrained vascular regeneration and increased preretinal neovascularization in Nrf2-deficient mice in OIR. ( A ) Retinal flat mounts show that intravitreal injection of Lv. shSema6A at P7 leads to a dramatic decreases

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Inhibition of Sema6A abrogates the restrained vascular regeneration and increased preretinal neovascularization in Nrf2-deficient mice in OIR. ( A ) Retinal flat mounts show that intravitreal injection of Lv. shSema6A at P7 leads to a dramatic decreases

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Inhibition, Mouse Assay, Injection

    ( A and B ) Nrf2 −/− retinal vasculature showed less perfusion compared with wild-type. More poor-perfused vessels were observed in Nrf2 −/− retinas (arrowheads) at P17. n = 7. (Scale bar, 50 µm.) ( C and D ) No significant

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: ( A and B ) Nrf2 −/− retinal vasculature showed less perfusion compared with wild-type. More poor-perfused vessels were observed in Nrf2 −/− retinas (arrowheads) at P17. n = 7. (Scale bar, 50 µm.) ( C and D ) No significant

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques:

    Deletion of the Nrf2 repressor Keap1 in ECs leads to a dramatic reduction in preretinal neovascularization and a modest enhancement in vessel regrowth in OIR. ( A ) Retinal flat mounts of control ( Keap1 fl/fl ) and Keap1 fl/fl ; Cdh5-Cre mice at P17 of OIR.

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Deletion of the Nrf2 repressor Keap1 in ECs leads to a dramatic reduction in preretinal neovascularization and a modest enhancement in vessel regrowth in OIR. ( A ) Retinal flat mounts of control ( Keap1 fl/fl ) and Keap1 fl/fl ; Cdh5-Cre mice at P17 of OIR.

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Mouse Assay

    Neuronal Nrf2 plays a crucial role in revascularization in OIR. ( A and B ) Nrf2 localization in retinal cryosections from P12 (2 h) OIR. Nrf2 is ubiquitously expressed in both avascular and vascularized retina, and strong expression was observed in GCL

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Neuronal Nrf2 plays a crucial role in revascularization in OIR. ( A and B ) Nrf2 localization in retinal cryosections from P12 (2 h) OIR. Nrf2 is ubiquitously expressed in both avascular and vascularized retina, and strong expression was observed in GCL

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Expressing

    Pharmacologic activation of Nrf2 by CDDO-Im has no effect on retinal revascularization in OIR in Nrf2 −/− mice. Nrf2 −/− mice were intravitreally injected with 1 µL 24 nM CDDO-Im twice at P12 and P14 of OIR. ( A and

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Pharmacologic activation of Nrf2 by CDDO-Im has no effect on retinal revascularization in OIR in Nrf2 −/− mice. Nrf2 −/− mice were intravitreally injected with 1 µL 24 nM CDDO-Im twice at P12 and P14 of OIR. ( A and

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Activation Assay, Mouse Assay, Injection

    ( A ) Quantitative RT-PCR analysis of Nrf2 in isolated primary RGCs from Nrf2 fl/fl and Nrf2 fl/fl ; Six3-Cre mice. ( B ) Quantitative RT-PCR analysis of Nrf2 and NQO1 in RGC-5 treated with control siRNA or Nrf2 siRNA for 36 h. ( C ) Quantitative RT-PCR analysis

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: ( A ) Quantitative RT-PCR analysis of Nrf2 in isolated primary RGCs from Nrf2 fl/fl and Nrf2 fl/fl ; Six3-Cre mice. ( B ) Quantitative RT-PCR analysis of Nrf2 and NQO1 in RGC-5 treated with control siRNA or Nrf2 siRNA for 36 h. ( C ) Quantitative RT-PCR analysis

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Quantitative RT-PCR, Isolation, Mouse Assay

    Hypoxia Accentuates Sema6A Expression in Nrf2-Deficient RGCs via Hypoxia-Inducible Factor-1 Alpha.

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Hypoxia Accentuates Sema6A Expression in Nrf2-Deficient RGCs via Hypoxia-Inducible Factor-1 Alpha.

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Expressing

    Genetic ablation of Nrf2 impedes vascular regrowth and increases pathological neovascularization in OIR. ( A ) Diagram of OIR. Neonatal mice are put into hyperoxia chamber containing 75% O 2 at P7 and return to room air at P12. The retinal blood vessels

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Genetic ablation of Nrf2 impedes vascular regrowth and increases pathological neovascularization in OIR. ( A ) Diagram of OIR. Neonatal mice are put into hyperoxia chamber containing 75% O 2 at P7 and return to room air at P12. The retinal blood vessels

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Mouse Assay

    Pharmacologic activation of Nrf2 by CDDO-Im improves retinal revascularization and reduces pathologic neovascularization. ( A ) Schematic of CDDO-Im administration in OIR. Wild-type mice were intravitreally injected with 1 µL 24 nM CDDO-Im twice

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Pharmacologic activation of Nrf2 by CDDO-Im improves retinal revascularization and reduces pathologic neovascularization. ( A ) Schematic of CDDO-Im administration in OIR. Wild-type mice were intravitreally injected with 1 µL 24 nM CDDO-Im twice

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Activation Assay, Mouse Assay, Injection

    Hypoxia accentuates expression of Sema6A in Nrf2-deficient RGCs via HIF-1α. ( A and B ) Quantitative RT-PCR analysis of Sema6A ( A ) and VEGF ( B ) in primary RGCs isolated from Nrf2 fl/fl and Nrf2 fl/fl ; Six3-Cre mice. Exposure to hypoxia for 4 h induces

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Hypoxia accentuates expression of Sema6A in Nrf2-deficient RGCs via HIF-1α. ( A and B ) Quantitative RT-PCR analysis of Sema6A ( A ) and VEGF ( B ) in primary RGCs isolated from Nrf2 fl/fl and Nrf2 fl/fl ; Six3-Cre mice. Exposure to hypoxia for 4 h induces

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay

    ( A ) Quantitative RT-PCR analysis of axon guidance genes in wild-type and Nrf2 −/− retinas at P15of OIR. n = 5. ( B ) Quantitative RT-PCR analysis shows the increase in Sema6A , whereas no change in Sema3A or Sema3E was seen in the central

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: ( A ) Quantitative RT-PCR analysis of axon guidance genes in wild-type and Nrf2 −/− retinas at P15of OIR. n = 5. ( B ) Quantitative RT-PCR analysis shows the increase in Sema6A , whereas no change in Sema3A or Sema3E was seen in the central

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Quantitative RT-PCR

    Deletion of Nrf2 leads to increased Sema6A expression in ischemic inner retina in OIR. ( A ) Quantitative RT-PCR analysis shows the increased Sema6A mRNA in Nrf2 −/− retinas compared with wild-type from P12 to P15 in OIR. n = 5. ( B ) Retinal

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Deletion of Nrf2 leads to increased Sema6A expression in ischemic inner retina in OIR. ( A ) Quantitative RT-PCR analysis shows the increased Sema6A mRNA in Nrf2 −/− retinas compared with wild-type from P12 to P15 in OIR. n = 5. ( B ) Retinal

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Expressing, Quantitative RT-PCR

    Deletion of Nrf2 Leads to Increased Sema6A Expression in Ischemic Inner Retina in OIR.

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Deletion of Nrf2 Leads to Increased Sema6A Expression in Ischemic Inner Retina in OIR.

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques: Expressing

    Schematic of principal findings depicting a crucial role of Nrf2 in promoting vascular regeneration in ischemic retinopathy. ( A ) Nrf2 is activated in EC and RGC during vascular regeneration of ischemic retinas. Loss of Nrf2 in EC leads to a mild decrease

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

    Article Title: Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    doi: 10.1073/pnas.1512683112

    Figure Lengend Snippet: Schematic of principal findings depicting a crucial role of Nrf2 in promoting vascular regeneration in ischemic retinopathy. ( A ) Nrf2 is activated in EC and RGC during vascular regeneration of ischemic retinas. Loss of Nrf2 in EC leads to a mild decrease

    Article Snippet: The following antibodies were used: monoclonal rabbit anti-Nrf2 (Abcam), polyclonal goat anti-Brn3 (Santa Cruz Biotechnology), monoclonal rat anti-PDGFRα (BD Biosciences), monoclonal rabbit anti-Vimentin (Sigma), polyclonal goat anti-Sema6A (R & D Systems), and monoclonal rabbit anti-Tuj1 (Covance).

    Techniques:

    Glutathione measurement and siRNA transfection in HepG2. Cells were incubated in the absence (control) or presence of DL-propargylglycine (PAG) for 16 h and total glutathione was measured (a). Cells were transfected with Nrf2 siRNA or scrambled siRNA (control). The mRNA of Nrf2 (b) and glutamate-cysteine ligase subunits Gclc (c) and Gclm (d) were determined. Results are expressed as mean ± SE ( n = 4 for each group). ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Downregulation of Glutathione Biosynthesis Contributes to Oxidative Stress and Liver Dysfunction in Acute Kidney Injury

    doi: 10.1155/2016/9707292

    Figure Lengend Snippet: Glutathione measurement and siRNA transfection in HepG2. Cells were incubated in the absence (control) or presence of DL-propargylglycine (PAG) for 16 h and total glutathione was measured (a). Cells were transfected with Nrf2 siRNA or scrambled siRNA (control). The mRNA of Nrf2 (b) and glutamate-cysteine ligase subunits Gclc (c) and Gclm (d) were determined. Results are expressed as mean ± SE ( n = 4 for each group). ∗ p

    Article Snippet: Nuclear proteins (90 μ g) were used to determine Nrf2 protein with anti-rabbit Nrf2 monoclonal antibodies (1 : 500, Abcam, Inc., Toronto, Canada).

    Techniques: Transfection, Incubation

    Expression of Nrf2 protein in the liver. The Nrf2 protein was determined by Western immunoblotting analysis of the liver nuclear fraction of rats subjected to renal IR or sham operation. Results are expressed as mean ± SE ( n = 4 for each group). ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Downregulation of Glutathione Biosynthesis Contributes to Oxidative Stress and Liver Dysfunction in Acute Kidney Injury

    doi: 10.1155/2016/9707292

    Figure Lengend Snippet: Expression of Nrf2 protein in the liver. The Nrf2 protein was determined by Western immunoblotting analysis of the liver nuclear fraction of rats subjected to renal IR or sham operation. Results are expressed as mean ± SE ( n = 4 for each group). ∗ p

    Article Snippet: Nuclear proteins (90 μ g) were used to determine Nrf2 protein with anti-rabbit Nrf2 monoclonal antibodies (1 : 500, Abcam, Inc., Toronto, Canada).

    Techniques: Expressing, Western Blot

    The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) exhibited an increased Nrf2 expression when evaluated 6 months after exposure to GWI-related chemicals and stress. Figures (A1–A4) illustrate examples of neuron specific nuclear antigen positive (NeuN+) hippocampal CA3 pyramidal neurons (green) displaying nuclear translocation of Nrf2 (red particles) in a naïve control animal (arrows in A1,A2 ) and in an animal exposed to GWI-related chemicals and stress (arrows in B1,B2 ). Note that nuclear translocation of Nrf2 is increased in the animal exposed to GWI-related chemicals and stress (B1,B2) . Bar charts in C1–C3 compare percentages of NeuN+ hippocampal neurons displaying nuclear Nrf2 (C1) , percentages of hippocampal neurons showing robust nuclear Nrf2 (C2) and the extent of activated Nrf2 measured through ELISA in the entire hippocampus between age-matched naïve control animals and animals exposed to GWI-related chemicals and stress ( n = 5–6/group). Scale bar, (A1,B1) , 10 μm; (A2,B2) , 5 μm. ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Chronic Oxidative Stress, Mitochondrial Dysfunction, Nrf2 Activation and Inflammation in the Hippocampus Accompany Heightened Systemic Inflammation and Oxidative Stress in an Animal Model of Gulf War Illness

    doi: 10.3389/fnmol.2017.00182

    Figure Lengend Snippet: The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) exhibited an increased Nrf2 expression when evaluated 6 months after exposure to GWI-related chemicals and stress. Figures (A1–A4) illustrate examples of neuron specific nuclear antigen positive (NeuN+) hippocampal CA3 pyramidal neurons (green) displaying nuclear translocation of Nrf2 (red particles) in a naïve control animal (arrows in A1,A2 ) and in an animal exposed to GWI-related chemicals and stress (arrows in B1,B2 ). Note that nuclear translocation of Nrf2 is increased in the animal exposed to GWI-related chemicals and stress (B1,B2) . Bar charts in C1–C3 compare percentages of NeuN+ hippocampal neurons displaying nuclear Nrf2 (C1) , percentages of hippocampal neurons showing robust nuclear Nrf2 (C2) and the extent of activated Nrf2 measured through ELISA in the entire hippocampus between age-matched naïve control animals and animals exposed to GWI-related chemicals and stress ( n = 5–6/group). Scale bar, (A1,B1) , 10 μm; (A2,B2) , 5 μm. ∗ p

    Article Snippet: In brief, sections were first processed for Nrf2 immunofluorescence, which comprised sequential incubation of sections in 10% donkey serum for 30 min and rabbit anti-Nrf2 solution (1:500; Abcam, Cambridge, MA, United States) overnight.

    Techniques: Expressing, Translocation Assay, Enzyme-linked Immunosorbent Assay

    Oxidative stress induces T reg cell apoptosis in the tumor environment. ( a ) The effect of ovarian cancer ascites on human T reg cell apoptosis. Mouse T reg cells and conventional T cells (T conv ) were cocultured with 50% human ovarian cancer ascites or hydrogen peroxide for 24 h. Additional cultures were treated with N -acetyl-cysteine (NAC) as a free radical scavenger. Annexin V + T reg cells and T conv cells were analyzed by flow cytometry. n = 5. ( b,c ) The effect of ovarian cancer ascites on proapoptotic and antiapoptotic gene transcripts in T reg cells. T reg cells were exposed to ascites or ascites plus NAC after 24 h. Proapoptotic ( b ) and antiapoptotic ( c ) transcripts were quantified by real-time PCR. n = 3. ( d,e ) The effect of hydrogen peroxide on proapoptotic and antiapoptotic transcripts in T reg cells. T reg cells were exposed to hydrogen peroxide or hydrogen peroxide plus NAC after 24 h. Proapoptotic ( d ) and antiapoptotic ( e ) mouse gene transcripts were quantified by real-time PCR. n = 3. ( f,g ) Expression of mouse NRF2 and NRF2-associated gene transcripts ( f ) and proteins ( g ) in T cell subsets as determined by real-time PCR and immunoblotting, respectively. n = 5. ( h ) The expression of intracellular ROS in T reg cells. T reg cells and conventional T cells were cultured overnight. Amounts of intracellular ROS were measured by flow cytometry. n = 5. MFI, mean fluorescence intensity. ( i ) The effect of NRF2 inducers on T reg cell apoptosis in vitro . Mouse T reg cells were cultured for 24 h with 50% ascites or medium containing H 2 O 2 . NRF2 inducers diethylmaleate (DEM; 100 μM) and sulforaphane (10 μM) were added to the culture for 24 h. Controls were treated with vehicle (DMSO). Apoptosis was measured by flow cytometry with annexin V. n = 4. ( j – l ) The effect of sulforaphane on tumor immunity. MC38-bearing mice were treated with sulforaphane (25 mg/kg, 5 d per week) or vehicle (DMSO). Tumor T reg cell apoptosis ( j ) and CD8 + T cell polyfunctional cytokine expression ( k ) were analyzed by flow cytometry. Tumor volume was monitored ( l ). n = 10. All data are shown as the mean and s.d. and are from 1 experiment with 10 animals per group ( i – l ) or from single experiments with cells isolated from individual animal for each data point ( a – h ). * P

    Journal: Nature immunology

    Article Title: Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor

    doi: 10.1038/ni.3868

    Figure Lengend Snippet: Oxidative stress induces T reg cell apoptosis in the tumor environment. ( a ) The effect of ovarian cancer ascites on human T reg cell apoptosis. Mouse T reg cells and conventional T cells (T conv ) were cocultured with 50% human ovarian cancer ascites or hydrogen peroxide for 24 h. Additional cultures were treated with N -acetyl-cysteine (NAC) as a free radical scavenger. Annexin V + T reg cells and T conv cells were analyzed by flow cytometry. n = 5. ( b,c ) The effect of ovarian cancer ascites on proapoptotic and antiapoptotic gene transcripts in T reg cells. T reg cells were exposed to ascites or ascites plus NAC after 24 h. Proapoptotic ( b ) and antiapoptotic ( c ) transcripts were quantified by real-time PCR. n = 3. ( d,e ) The effect of hydrogen peroxide on proapoptotic and antiapoptotic transcripts in T reg cells. T reg cells were exposed to hydrogen peroxide or hydrogen peroxide plus NAC after 24 h. Proapoptotic ( d ) and antiapoptotic ( e ) mouse gene transcripts were quantified by real-time PCR. n = 3. ( f,g ) Expression of mouse NRF2 and NRF2-associated gene transcripts ( f ) and proteins ( g ) in T cell subsets as determined by real-time PCR and immunoblotting, respectively. n = 5. ( h ) The expression of intracellular ROS in T reg cells. T reg cells and conventional T cells were cultured overnight. Amounts of intracellular ROS were measured by flow cytometry. n = 5. MFI, mean fluorescence intensity. ( i ) The effect of NRF2 inducers on T reg cell apoptosis in vitro . Mouse T reg cells were cultured for 24 h with 50% ascites or medium containing H 2 O 2 . NRF2 inducers diethylmaleate (DEM; 100 μM) and sulforaphane (10 μM) were added to the culture for 24 h. Controls were treated with vehicle (DMSO). Apoptosis was measured by flow cytometry with annexin V. n = 4. ( j – l ) The effect of sulforaphane on tumor immunity. MC38-bearing mice were treated with sulforaphane (25 mg/kg, 5 d per week) or vehicle (DMSO). Tumor T reg cell apoptosis ( j ) and CD8 + T cell polyfunctional cytokine expression ( k ) were analyzed by flow cytometry. Tumor volume was monitored ( l ). n = 10. All data are shown as the mean and s.d. and are from 1 experiment with 10 animals per group ( i – l ) or from single experiments with cells isolated from individual animal for each data point ( a – h ). * P

    Article Snippet: Then they were analyzed by SDS–PAGE followed by western blotting with primary antibodies as follows: anti-human/mouse NRF2 (clone 1808Y; Abcam), anti-human/mouse GCLM (rabbit polyclonal; Abcam), anti-mouse/human PD1 (clone 7A11B1; Thermo Fisher Scientific), anti-mouse PD-L1 (goat polyclonal; R & D Systems), anti-mouse CTLA-4 (clone 63828; R & D Systems), anti-mouse CD39 (clone 495826; R & D Systems), and anti-mouse CD73 (clone sc-32299; Santa Cruz Biotechnology).

    Techniques: Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Fluorescence, In Vitro, Mouse Assay, Isolation

    Detection of NRF2 and Keap1 protein expression assessed by immunohistochemical staining in representative specimens of normal cervical epithelia, CIN and CSCC, respectively. A: Expression of NRF2 in normal cervical epithelia with weak cytoplasm staining; B: Moderate expression of NRF2 protein in CIN tissue; C: Strong nucleus expression of NRF2 proteins in CSCC tissue; D: Expression of Keap1 in normal cervical epithelia with Strong cytoplasm staining; E: Moderate expression of Keap1 protein in CIN tissue; F: weak expression of Keap1 proteins in CSCC tissue. (original magnification, × 200).

    Journal: PLoS ONE

    Article Title: Functional Role of NRF2 in Cervical Carcinogenesis

    doi: 10.1371/journal.pone.0133876

    Figure Lengend Snippet: Detection of NRF2 and Keap1 protein expression assessed by immunohistochemical staining in representative specimens of normal cervical epithelia, CIN and CSCC, respectively. A: Expression of NRF2 in normal cervical epithelia with weak cytoplasm staining; B: Moderate expression of NRF2 protein in CIN tissue; C: Strong nucleus expression of NRF2 proteins in CSCC tissue; D: Expression of Keap1 in normal cervical epithelia with Strong cytoplasm staining; E: Moderate expression of Keap1 protein in CIN tissue; F: weak expression of Keap1 proteins in CSCC tissue. (original magnification, × 200).

    Article Snippet: Immunohistochemistry (IHC) IHC staining was performed with an anti-Keap1 rat monoclonal antibody (1:5,000) and an anti-human NRF2 mouse monoclonal antibody (1:300 Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Immunohistochemistry, Staining

    NRF2 positively modulates CSCC cellular malignant phenotypes. A, D, G and J: Cell apoptosis, Proliferation, Migration and invasion in Siha cells, respectively (Normal controls). B, E, H and K: a Knockdown of NRF2 increased cell apoptosis, decreased cell proliferation, migration and invasion, which significantly decreased malignant phenotypes of Siha cells. C, F, I and L: Overexpression of NRF2 sharply decreased cell apoptosis, increased cell proliferation, migration and invasion, which significantly enhanced cell proliferation and migration in Siha cell line. All experiments were performed at least three times.

    Journal: PLoS ONE

    Article Title: Functional Role of NRF2 in Cervical Carcinogenesis

    doi: 10.1371/journal.pone.0133876

    Figure Lengend Snippet: NRF2 positively modulates CSCC cellular malignant phenotypes. A, D, G and J: Cell apoptosis, Proliferation, Migration and invasion in Siha cells, respectively (Normal controls). B, E, H and K: a Knockdown of NRF2 increased cell apoptosis, decreased cell proliferation, migration and invasion, which significantly decreased malignant phenotypes of Siha cells. C, F, I and L: Overexpression of NRF2 sharply decreased cell apoptosis, increased cell proliferation, migration and invasion, which significantly enhanced cell proliferation and migration in Siha cell line. All experiments were performed at least three times.

    Article Snippet: Immunohistochemistry (IHC) IHC staining was performed with an anti-Keap1 rat monoclonal antibody (1:5,000) and an anti-human NRF2 mouse monoclonal antibody (1:300 Abcam, Cambridge, MA, USA).

    Techniques: Migration, Over Expression

    Effect of siRNA-expressing vectors and over-expressing vectors on the cell migration and invasion of Siha cells following transfection. For overexpression studies, NRF2 was overexpressed using a pcDNA3.1 vector and inhibition of NRF2 was achieved using siRNA vectors. A and B: regulates migration of Siha cells; C and D: regulates invasion of Siha cells. * P

    Journal: PLoS ONE

    Article Title: Functional Role of NRF2 in Cervical Carcinogenesis

    doi: 10.1371/journal.pone.0133876

    Figure Lengend Snippet: Effect of siRNA-expressing vectors and over-expressing vectors on the cell migration and invasion of Siha cells following transfection. For overexpression studies, NRF2 was overexpressed using a pcDNA3.1 vector and inhibition of NRF2 was achieved using siRNA vectors. A and B: regulates migration of Siha cells; C and D: regulates invasion of Siha cells. * P

    Article Snippet: Immunohistochemistry (IHC) IHC staining was performed with an anti-Keap1 rat monoclonal antibody (1:5,000) and an anti-human NRF2 mouse monoclonal antibody (1:300 Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Migration, Transfection, Over Expression, Plasmid Preparation, Inhibition

    The detection of NRF2 protein transfection with mimics and inhibitor. Morphology of transfected Siha cells for 48 h under microscopy (magnification ×200). A. Transfection with Short interfering RNA (SiRNA); B. transfection with mimics; C. The levels of NRF2 protein detected by Western blotting after transfection for 72 h.1 and 2 were normal control; 3 and 4 were Knockdown group; 5 and 6 were normal control; 7 and 8 were overexpression group; D The relative expression of NRF2 was displayed, which normalized to b-tubulin. There is a statistically significant difference between the group transfected with NRF2 mimics, NRF2 inhibitor and normal control.

    Journal: PLoS ONE

    Article Title: Functional Role of NRF2 in Cervical Carcinogenesis

    doi: 10.1371/journal.pone.0133876

    Figure Lengend Snippet: The detection of NRF2 protein transfection with mimics and inhibitor. Morphology of transfected Siha cells for 48 h under microscopy (magnification ×200). A. Transfection with Short interfering RNA (SiRNA); B. transfection with mimics; C. The levels of NRF2 protein detected by Western blotting after transfection for 72 h.1 and 2 were normal control; 3 and 4 were Knockdown group; 5 and 6 were normal control; 7 and 8 were overexpression group; D The relative expression of NRF2 was displayed, which normalized to b-tubulin. There is a statistically significant difference between the group transfected with NRF2 mimics, NRF2 inhibitor and normal control.

    Article Snippet: Immunohistochemistry (IHC) IHC staining was performed with an anti-Keap1 rat monoclonal antibody (1:5,000) and an anti-human NRF2 mouse monoclonal antibody (1:300 Abcam, Cambridge, MA, USA).

    Techniques: Transfection, Microscopy, Small Interfering RNA, Western Blot, Over Expression, Expressing

    Keap-1 and Nrf2 expression in different tissues measured via western blot analysis. The expression of Keap-1 and Nrf2 in the (A) heart, (B) lung and (C) kidney tissue. All data are presented as the mean ± standard deviation and were obtained from at least six independent experiments or tests. 1, Normal control group; 2, Normal obese group; 3, Normal asthma group; 4, Obese + asthma group; 5, RSV obese group; 6, RSV asthma group; 7, RSV obese + asthma group; RSV, resveratrol; Keap-1, kelch-like ECH associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Resveratrol protects against oxidative stress by activating the Keap-1/Nrf2 antioxidant defense system in obese-asthmatic rats

    doi: 10.3892/etm.2018.6747

    Figure Lengend Snippet: Keap-1 and Nrf2 expression in different tissues measured via western blot analysis. The expression of Keap-1 and Nrf2 in the (A) heart, (B) lung and (C) kidney tissue. All data are presented as the mean ± standard deviation and were obtained from at least six independent experiments or tests. 1, Normal control group; 2, Normal obese group; 3, Normal asthma group; 4, Obese + asthma group; 5, RSV obese group; 6, RSV asthma group; 7, RSV obese + asthma group; RSV, resveratrol; Keap-1, kelch-like ECH associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2.

    Article Snippet: Subsequently, membranes were incubated with rabbit anti-rat Keap-1 polyclonal antibody (1:3,000; cat. no. ab139729), rabbit anti-rat Nrf2 monoclonal antibody (1:3,000; cat. no. ab181602) and rabbit anti-rat GAPDH polyclonal antibody (1:2,000; cat. no. ab9485) (all Abcam, Cambridge, MA, USA) at room temperature for 2 h. Membranes were washed using PBST and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2,000; cat. no. A0545; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

    Techniques: Expressing, Western Blot, Standard Deviation

    Ferulic acid induces Nrf2 activation and translocation into the nucleus. (A–D) Representative images from four independent immunofluorescence experiments in which we performed a double-labeling with DAPI (A 1 –D 1 ) and an anti-Nrf2 antibody (A 2 –D 2 ) . Merged images are shown in (A 3 –D 3 ) . XZ and YZ cross-sections in the boxes (referred to the dashed lines) from the confocal Z -stack acquisitions illustrate cytosolic or nuclear fluorescence signal(s) (XZ and YZ boxes: a 1 –d 1 refer to DAPI staining; a 2 –d 2 : refer to Nrf2 fluorescence; a 3 –d 3 : Merge). In cells treated with TMT, a strong Nrf2 activation was detected which, however, remains mainly confined in the cytoplasm (b 1 –b 3 in B) . In TMT + FA treated cells, there was a further cytoplasmic enhancement of Nrf2 expression compared to TMT alone which also translocates into the nucleus as indicated by Z -stack acquisitions (c 1 –c 3 in C) . FA administration (10 μM) induced an endogenous antioxidant response leading to a strong increase in Nrf2 expression in the cytosol (d 1 –d 3 in D) compared to Control (a 1 -a 3 in A) . Scale bar: 20 μm. For further information see text.

    Journal: Frontiers in Pharmacology

    Article Title: Ferulic Acid Regulates the Nrf2/Heme Oxygenase-1 System and Counteracts Trimethyltin-Induced Neuronal Damage in the Human Neuroblastoma Cell Line SH-SY5Y

    doi: 10.3389/fphar.2015.00305

    Figure Lengend Snippet: Ferulic acid induces Nrf2 activation and translocation into the nucleus. (A–D) Representative images from four independent immunofluorescence experiments in which we performed a double-labeling with DAPI (A 1 –D 1 ) and an anti-Nrf2 antibody (A 2 –D 2 ) . Merged images are shown in (A 3 –D 3 ) . XZ and YZ cross-sections in the boxes (referred to the dashed lines) from the confocal Z -stack acquisitions illustrate cytosolic or nuclear fluorescence signal(s) (XZ and YZ boxes: a 1 –d 1 refer to DAPI staining; a 2 –d 2 : refer to Nrf2 fluorescence; a 3 –d 3 : Merge). In cells treated with TMT, a strong Nrf2 activation was detected which, however, remains mainly confined in the cytoplasm (b 1 –b 3 in B) . In TMT + FA treated cells, there was a further cytoplasmic enhancement of Nrf2 expression compared to TMT alone which also translocates into the nucleus as indicated by Z -stack acquisitions (c 1 –c 3 in C) . FA administration (10 μM) induced an endogenous antioxidant response leading to a strong increase in Nrf2 expression in the cytosol (d 1 –d 3 in D) compared to Control (a 1 -a 3 in A) . Scale bar: 20 μm. For further information see text.

    Article Snippet: Samples were then incubated for 3 h with primary rabbit anti-4-HNE (Cat#HNE11-S, Alpha Diagnostic Int., San Antonio, TX, USA) or mouse anti-Nrf2 (Abcam, Cambridge, UK) and rabbit anti-HO-1 (Stressgen, Ann Arbor, MI, USA) antibodies diluted 1:100 in 0.3% BSA in PBS.

    Techniques: Activation Assay, Translocation Assay, Immunofluorescence, Labeling, Fluorescence, Staining, Expressing

    ABL1 coordinates NRF2 antioxidant response in vitro

    Journal: Cancer cell

    Article Title: Targeting ABL1-mediated Oxidative Stress Adaptation in Fumarate Hydratase-Deficient Cancer

    doi: 10.1016/j.ccell.2014.10.005

    Figure Lengend Snippet: ABL1 coordinates NRF2 antioxidant response in vitro

    Article Snippet: Mouse anti-NRF2 antibody (dilution 1:100; dilution; Abcam # 62352) was added and chamber slides were incubated overnight at 4°C in a humidified atmosphere.

    Techniques: In Vitro

    Silencing Nrf2 RNA skews macrophages toward the M2 phenotype NR8383 cells were transfected with either silencing RNA to Nrf2 (si-Nrf2) or with a control (scrambled) silencing RNA (si-Ctrl). Twenty-four hours after transfection, gene expression of Arg1 and iNOS were assessed by RT-PCR. Cells transfected with si- Nrf2 were significantly skewed toward the M2 phenotype, as shown by an elevated Arg1/iNOS ratio. Data presented as a ratio of fold change, mean ± SEM, n=5, *p

    Journal: Journal of clinical & cellular immunology

    Article Title: Activation of Alveolar Macrophages with Interferon-γ Promotes Antioxidant Defenses via the Nrf2-ARE Pathway

    doi: 10.4172/2155-9899.1000365

    Figure Lengend Snippet: Silencing Nrf2 RNA skews macrophages toward the M2 phenotype NR8383 cells were transfected with either silencing RNA to Nrf2 (si-Nrf2) or with a control (scrambled) silencing RNA (si-Ctrl). Twenty-four hours after transfection, gene expression of Arg1 and iNOS were assessed by RT-PCR. Cells transfected with si- Nrf2 were significantly skewed toward the M2 phenotype, as shown by an elevated Arg1/iNOS ratio. Data presented as a ratio of fold change, mean ± SEM, n=5, *p

    Article Snippet: Cells were stained with the following antibodies: anti-Nrf2 mouse monoclonal antibody (Abcam, Cambridge, MA, USA), anti-GCLC rabbit polyclonal antibody (Abcam), anti-mouse Pacific Blue (Life Technologies), and anti-rabbit Alexa Fluor 555 (Life Technologies).

    Techniques: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction

    Nrf2-ARE activity is enhanced in cells polarized with IFN- γ NR8383 cells (1 million/well) were treated with IFN-γ as above. Seventy-two hours later, protein expression of Nrf2 and its downstream effector GCLC were significantly increased in IFN-γ polarized cells as assessed by flow cytometry. Data presented as mean ± SEM, n=3. *p

    Journal: Journal of clinical & cellular immunology

    Article Title: Activation of Alveolar Macrophages with Interferon-γ Promotes Antioxidant Defenses via the Nrf2-ARE Pathway

    doi: 10.4172/2155-9899.1000365

    Figure Lengend Snippet: Nrf2-ARE activity is enhanced in cells polarized with IFN- γ NR8383 cells (1 million/well) were treated with IFN-γ as above. Seventy-two hours later, protein expression of Nrf2 and its downstream effector GCLC were significantly increased in IFN-γ polarized cells as assessed by flow cytometry. Data presented as mean ± SEM, n=3. *p

    Article Snippet: Cells were stained with the following antibodies: anti-Nrf2 mouse monoclonal antibody (Abcam, Cambridge, MA, USA), anti-GCLC rabbit polyclonal antibody (Abcam), anti-mouse Pacific Blue (Life Technologies), and anti-rabbit Alexa Fluor 555 (Life Technologies).

    Techniques: Activity Assay, Expressing, Flow Cytometry, Cytometry

    Silencing Nrf2 RNA abrogates the enhanced response to oxidative stress seen in IFN-γ treated cells NR8383 cells were polarized with either IFN-γ for 48 hours. The cells were then transfected with either silencing RNA to Nrf2 (si-Nrf2) or with a control (scrambled) silencing RNA (si-Ctrl). After transfection, cells were again plated in media containing IFN-γ. Twenty-four hours post-transfection (seventy-two hours after polarization), cells were treated with GOX (5 mU/ml). After four hours, remaining H 2 O 2 concentration was assessed by Amplex Red Assay. Cells transfected with si-Nrf2 were significantly worse at scavenging hydrogen peroxide when compared to those transfected with si-Ctrl.

    Journal: Journal of clinical & cellular immunology

    Article Title: Activation of Alveolar Macrophages with Interferon-γ Promotes Antioxidant Defenses via the Nrf2-ARE Pathway

    doi: 10.4172/2155-9899.1000365

    Figure Lengend Snippet: Silencing Nrf2 RNA abrogates the enhanced response to oxidative stress seen in IFN-γ treated cells NR8383 cells were polarized with either IFN-γ for 48 hours. The cells were then transfected with either silencing RNA to Nrf2 (si-Nrf2) or with a control (scrambled) silencing RNA (si-Ctrl). After transfection, cells were again plated in media containing IFN-γ. Twenty-four hours post-transfection (seventy-two hours after polarization), cells were treated with GOX (5 mU/ml). After four hours, remaining H 2 O 2 concentration was assessed by Amplex Red Assay. Cells transfected with si-Nrf2 were significantly worse at scavenging hydrogen peroxide when compared to those transfected with si-Ctrl.

    Article Snippet: Cells were stained with the following antibodies: anti-Nrf2 mouse monoclonal antibody (Abcam, Cambridge, MA, USA), anti-GCLC rabbit polyclonal antibody (Abcam), anti-mouse Pacific Blue (Life Technologies), and anti-rabbit Alexa Fluor 555 (Life Technologies).

    Techniques: Transfection, Concentration Assay, Amplex Red Assay

    Effects of ferulic acid (FA) and dimethylbiguanide (DMBG) on Keap-1 and Nrf2 expression in hepatocytes and cardiomyocytes. (a) Western blot results. (b) Keap-1 and Nrf2 expression levels in hepatocytes. (c) Keap-1 and Nrf2 expression levels in cardiomyocytes. Mean±SEM, n =3. ## p

    Journal: Food & Nutrition Research

    Article Title: Cytoprotective mechanism of ferulic acid against high glucose-induced oxidative stress in cardiomyocytes and hepatocytes

    doi: 10.3402/fnr.v60.30323

    Figure Lengend Snippet: Effects of ferulic acid (FA) and dimethylbiguanide (DMBG) on Keap-1 and Nrf2 expression in hepatocytes and cardiomyocytes. (a) Western blot results. (b) Keap-1 and Nrf2 expression levels in hepatocytes. (c) Keap-1 and Nrf2 expression levels in cardiomyocytes. Mean±SEM, n =3. ## p

    Article Snippet: The membranes were blocked at room temperature for 2 h with BSA and incubated at 4°C overnight using relative primary antibodies at 1:1,000: rabbit anti-rat Keap1 antibody (CST, Boston, MA, USA), rabbit anti-rat Nrf2 antibody (Abcam), rabbit anti-rat GAPDH antibody (CST), and rabbit anti-rat histone H3 antibody (CST).

    Techniques: Expressing, Western Blot

    SFN exerts its renoprotective role via the activation of Nrf2 in HK2 cells. (a) Cells were treated for 48 h with control or Nrf2 siRNA (the transfection efficiency was measured by western blot analysis). (b) SFN did not increase the Nrf2 nuclear protein level in Nrf2-deficient cells. (c) SFN did not increase the HO-1 protein level in Nrf2-deficient cells. (d) SFN did not increase cell viability in Nrf2-deficient cells. (e) SFN did not decrease reactive oxygen species in Nrf2-deficient cells. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sulforaphane Attenuates Contrast-Induced Nephropathy in Rats via Nrf2/HO-1 Pathway

    doi: 10.1155/2016/9825623

    Figure Lengend Snippet: SFN exerts its renoprotective role via the activation of Nrf2 in HK2 cells. (a) Cells were treated for 48 h with control or Nrf2 siRNA (the transfection efficiency was measured by western blot analysis). (b) SFN did not increase the Nrf2 nuclear protein level in Nrf2-deficient cells. (c) SFN did not increase the HO-1 protein level in Nrf2-deficient cells. (d) SFN did not increase cell viability in Nrf2-deficient cells. (e) SFN did not decrease reactive oxygen species in Nrf2-deficient cells. ∗ P

    Article Snippet: Following microwave treatment, the slides were incubated with the rabbit anti-mouse Nrf2 antibody (Abcam) at 4°C overnight.

    Techniques: Activation Assay, Transfection, Western Blot

    Semiquantitative analysis of Nrf2 and HO-1 immunoactivities in the kidneys of different groups. (a) Nrf2, (b) HO-1. Data are presented as the means ± SE ( n = 6). ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sulforaphane Attenuates Contrast-Induced Nephropathy in Rats via Nrf2/HO-1 Pathway

    doi: 10.1155/2016/9825623

    Figure Lengend Snippet: Semiquantitative analysis of Nrf2 and HO-1 immunoactivities in the kidneys of different groups. (a) Nrf2, (b) HO-1. Data are presented as the means ± SE ( n = 6). ∗ P

    Article Snippet: Following microwave treatment, the slides were incubated with the rabbit anti-mouse Nrf2 antibody (Abcam) at 4°C overnight.

    Techniques:

    SFN increased the expression levels of Nrf2, HO-1, and NQO-1 in HK2 cells after Ioversol exposure. (a) The relative expression of Nrf2; (b) the relative expression of HO-1; and (c) the relative expression of NQO-1. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sulforaphane Attenuates Contrast-Induced Nephropathy in Rats via Nrf2/HO-1 Pathway

    doi: 10.1155/2016/9825623

    Figure Lengend Snippet: SFN increased the expression levels of Nrf2, HO-1, and NQO-1 in HK2 cells after Ioversol exposure. (a) The relative expression of Nrf2; (b) the relative expression of HO-1; and (c) the relative expression of NQO-1. ∗ P

    Article Snippet: Following microwave treatment, the slides were incubated with the rabbit anti-mouse Nrf2 antibody (Abcam) at 4°C overnight.

    Techniques: Expressing

    SFN increased the expression levels of Nrf2, HO-1, and NQO-1 in CIN rats. (a) The relative expression of Nrf2; (b) the relative expression of HO-1; and (c) the relative expression of NQO-1. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sulforaphane Attenuates Contrast-Induced Nephropathy in Rats via Nrf2/HO-1 Pathway

    doi: 10.1155/2016/9825623

    Figure Lengend Snippet: SFN increased the expression levels of Nrf2, HO-1, and NQO-1 in CIN rats. (a) The relative expression of Nrf2; (b) the relative expression of HO-1; and (c) the relative expression of NQO-1. ∗ P

    Article Snippet: Following microwave treatment, the slides were incubated with the rabbit anti-mouse Nrf2 antibody (Abcam) at 4°C overnight.

    Techniques: Expressing

    The protein levels in different groups. (a) SFN pretreatment enhanced Nrf2 nuclear translocation. (b) Nrf2 protein levels in the cytoplasm. (c) SFN pretreatment increased NQO-1 and HO-1 protein levels in CIN rats.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sulforaphane Attenuates Contrast-Induced Nephropathy in Rats via Nrf2/HO-1 Pathway

    doi: 10.1155/2016/9825623

    Figure Lengend Snippet: The protein levels in different groups. (a) SFN pretreatment enhanced Nrf2 nuclear translocation. (b) Nrf2 protein levels in the cytoplasm. (c) SFN pretreatment increased NQO-1 and HO-1 protein levels in CIN rats.

    Article Snippet: Following microwave treatment, the slides were incubated with the rabbit anti-mouse Nrf2 antibody (Abcam) at 4°C overnight.

    Techniques: Translocation Assay

    Immunohistochemical photograph of Nrf2 in the kidneys of different groups. Original magnification ×400. (a) Control group; (b) Ioversol group; (c) Ioversol + SFN group; and (d) SFN group.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sulforaphane Attenuates Contrast-Induced Nephropathy in Rats via Nrf2/HO-1 Pathway

    doi: 10.1155/2016/9825623

    Figure Lengend Snippet: Immunohistochemical photograph of Nrf2 in the kidneys of different groups. Original magnification ×400. (a) Control group; (b) Ioversol group; (c) Ioversol + SFN group; and (d) SFN group.

    Article Snippet: Following microwave treatment, the slides were incubated with the rabbit anti-mouse Nrf2 antibody (Abcam) at 4°C overnight.

    Techniques: Immunohistochemistry

    BARD increases renal Nrf2 ( A ), PPARγ ( B ), and HO-1 ( C ) mRNA abundance after IR: densitometries. The y -axis shows the ratio of densitometry of the indicated gene to the densitometry of GAPDH. IR, ischemia caused by clamping the renal arteries,

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: BARD increases renal Nrf2 ( A ), PPARγ ( B ), and HO-1 ( C ) mRNA abundance after IR: densitometries. The y -axis shows the ratio of densitometry of the indicated gene to the densitometry of GAPDH. IR, ischemia caused by clamping the renal arteries,

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques:

    BARD increases renal Nrf2, PPARγ, and HO-1 mRNA abundance in surgically unmanipulated kidneys. Shown is a representative RT-PCR gel. 3H and 6H indicate 3 and 6 h after BARD or vehicle administration, respectively.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: BARD increases renal Nrf2, PPARγ, and HO-1 mRNA abundance in surgically unmanipulated kidneys. Shown is a representative RT-PCR gel. 3H and 6H indicate 3 and 6 h after BARD or vehicle administration, respectively.

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    BARD increases renal Nrf2 protein. Sections were immunostained for Nrf2 protein. A : semiquantitative analysis of the glomeruli. B : semiquantitative analysis of the peritubular capillaries of the cortex. The y -axis shows the number of Nrf2-positive cells

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: BARD increases renal Nrf2 protein. Sections were immunostained for Nrf2 protein. A : semiquantitative analysis of the glomeruli. B : semiquantitative analysis of the peritubular capillaries of the cortex. The y -axis shows the number of Nrf2-positive cells

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques:

    Semiquantitative analysis of renal HO-1 protein. Sections were immunostained for HO-1. The y -axis shows the number of Nrf2-positive cells per hpf. The statistical analyses for each time point were by ANOVA, and the comparisons between the indicated groups

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: Semiquantitative analysis of renal HO-1 protein. Sections were immunostained for HO-1. The y -axis shows the number of Nrf2-positive cells per hpf. The statistical analyses for each time point were by ANOVA, and the comparisons between the indicated groups

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques:

    Increased Nrf2 in BARD- vs. vehicle-treated ischemic renal cortex. A : anti-Nrf2 immunoperoxidase staining of BARD-treated ischemic kidneys at 8-h reperfusion. G, intensely Nrf2-positive glomeruli; T, one of many Nrf2-negative tubules; b, one rare Nrf2-positive

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: Increased Nrf2 in BARD- vs. vehicle-treated ischemic renal cortex. A : anti-Nrf2 immunoperoxidase staining of BARD-treated ischemic kidneys at 8-h reperfusion. G, intensely Nrf2-positive glomeruli; T, one of many Nrf2-negative tubules; b, one rare Nrf2-positive

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques: Immunoperoxidase Staining

    Localization of Nrf2 to glomerular endothelia of BARD-treated ischemic kidneys at 8-h reperfusion. Anti-CD31 is shown in green, anti-Nrf2 in red, and the overlap of both antibodies in yellow.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: Localization of Nrf2 to glomerular endothelia of BARD-treated ischemic kidneys at 8-h reperfusion. Anti-CD31 is shown in green, anti-Nrf2 in red, and the overlap of both antibodies in yellow.

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques:

    Localization of Nrf2 to cortical peritubular endothelia of BARD-treated ischemic kidneys at 8-h reperfusion. White arrows show a few of the many endothelia stained with anti-CD31 (green), anti-Nrf2 (red), and both antibodies (yellow).

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: Localization of Nrf2 to cortical peritubular endothelia of BARD-treated ischemic kidneys at 8-h reperfusion. White arrows show a few of the many endothelia stained with anti-CD31 (green), anti-Nrf2 (red), and both antibodies (yellow).

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques: Staining

    Semiquantitative analysis of renal PPARγ protein in glomeruli. Sections were immunostained for PPARγ protein. The y -axis shows the number of Nrf2-positive cells per hpf. The statistical analyses for each time point were by ANOVA, and the

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPAR?, and HO-1

    doi: 10.1152/ajprenal.00353.2010

    Figure Lengend Snippet: Semiquantitative analysis of renal PPARγ protein in glomeruli. Sections were immunostained for PPARγ protein. The y -axis shows the number of Nrf2-positive cells per hpf. The statistical analyses for each time point were by ANOVA, and the

    Article Snippet: For CD31 and Nrf2 Double staining, rat anti-mouse CD31 mAb (BD Biosciences Pharmingen) and rabbit anti-mouse Nrf2 mAb (Abcam) were used as the primary antibodies overnight at +4°C.

    Techniques:

    Trehalose inhibited H 2 O 2 -induced increase of intracellular ROS. (A) Statistical analysis showed that trehalose pretreatment prevented H 2 O 2 -induced increase of intracellular ROS. (B) Western blotting demonstrated that trehalose did not inhibit the upregulated expression of Nrf2 and phosphorylated Nrf2 caused by H 2 O 2 . (C and D)Enzyme activity assay revealed that trehalose suppressed H 2 O 2 -induced reduction in CAT and SOD. The values are expressed as mean±SEM (n=5 per group). *: p

    Journal: International Journal of Medical Sciences

    Article Title: Trehalose inhibits H2O2-induced autophagic death in dopaminergic SH-SY5Y cells via mitigation of ROS-dependent endoplasmic reticulum stress and AMPK activation

    doi: 10.7150/ijms.25656

    Figure Lengend Snippet: Trehalose inhibited H 2 O 2 -induced increase of intracellular ROS. (A) Statistical analysis showed that trehalose pretreatment prevented H 2 O 2 -induced increase of intracellular ROS. (B) Western blotting demonstrated that trehalose did not inhibit the upregulated expression of Nrf2 and phosphorylated Nrf2 caused by H 2 O 2 . (C and D)Enzyme activity assay revealed that trehalose suppressed H 2 O 2 -induced reduction in CAT and SOD. The values are expressed as mean±SEM (n=5 per group). *: p

    Article Snippet: Membranes were blocked with 3% bovine serum albumin in TBS for 30 min at room temperature, then incubated overnight at 4 ºC with the following primary antibodies including: rabbit polyclonal autophagy LC3 (1:1000; Sigma-Aldrich, St. Louis, MO), rabbit polyclonal Beclin-1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-p62(1:1000, Abcam, Cambridge, MA), rabbit monoclonal anti-ATG5(1:1000, Abcam, Cambridge, MA); rabbit polycolonal anti-GRP 78(1:1000, Abcam, Cambridge, MA), rabbit polyclonal anti-IRE-1(1:1000, Abcam, Cambridge, MA), rabbit polyclonal anti-ATF6(1:1000, Abcam, Cambridge, MA), rabbit monoclonal anti-Nrf2 (1:1000, Abcam, Cambridge, MA), rabbit monoclonal anti-phospho-Nrf2 (1:1000, Abcam, Cambridge, MA), rabbit polyclonal anti-PERK (1:1000, Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-phospho-PERK (1:1000, Cell Signaling Technology, Danvers, MA), and mouse monoclonal β-actin (1:2000; Santa Cruz Biotechnology).

    Techniques: Western Blot, Expressing, Activity Assay

    SFN up-regulates the expression and function of Nrf2 in the heart. Mice were treated as described in Fig. 1 . Nrf2 expression at mRNA and protein levels was detected by real-time PCR ( A ) and Western blot ( B ), respectively. The activation of Nrf2 was examined by immunohistochemical staining ( C , counter stain with hematoxylin, bar = 25μm) and Western blot ( D ) for Nrf2 phosphorylation at Ser40 (p-Nrf2). Nrf2 function was measured by determining the expression of Nrf2 downstream genes, catalase (CAT), NAD(P)H: quinone oxidoreductase (NQO1), and heme oxygenase 1 (HO-1), at both mRNA ( E ) and protein ( F ) levels with real-time PCR and Western blot, respectively. Data are presented as the mean ± SD (n = 7). *, p

    Journal: Redox Biology

    Article Title: Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway

    doi: 10.1016/j.redox.2017.12.016

    Figure Lengend Snippet: SFN up-regulates the expression and function of Nrf2 in the heart. Mice were treated as described in Fig. 1 . Nrf2 expression at mRNA and protein levels was detected by real-time PCR ( A ) and Western blot ( B ), respectively. The activation of Nrf2 was examined by immunohistochemical staining ( C , counter stain with hematoxylin, bar = 25μm) and Western blot ( D ) for Nrf2 phosphorylation at Ser40 (p-Nrf2). Nrf2 function was measured by determining the expression of Nrf2 downstream genes, catalase (CAT), NAD(P)H: quinone oxidoreductase (NQO1), and heme oxygenase 1 (HO-1), at both mRNA ( E ) and protein ( F ) levels with real-time PCR and Western blot, respectively. Data are presented as the mean ± SD (n = 7). *, p

    Article Snippet: The primary antibodies also included those against phosphorylated Akt (Ser473, p-Akt), phosphorylated GSK-3β (Ser9, p-GSK-3β), Akt, GSK-3β, and Fyn (Cell Signaling Technology, Danvers, MA, 1:1000 dilution), as well as 3-nitrotyrosine (3-NT, 1:3000 dilution, Millipore, Billerica, MA), 4-hydroxy-2-nonenal (4-HNE, 1:4000, Alpha Diagnostic International, San Antonio, TX), plasminogen activator inhibitor-1 (PAI-1, 1:2000, BD Biosciences, San Jose, CA), AT1 (1:1000, Abcam, Cambridge, MA), and phosphorylated Nrf2 at Ser40 antibody (p-Nrf2; 1:10,00, Abcam, Cambridge, MA).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Activation Assay, Immunohistochemistry, Staining

    Effects of genistein on GPR30 expression and reactive oxygen species (ROS) generation. (A) MDA-MB-231 and HCC1937 cells were treated with 50 μM Genistein. After 48 hours of incubation, the mRNA expression of GPR30 was assessed by reverse transcription-PCR. (B) The intracellular ROS levels in genistein treated MDA-MB-231 and HCC1937 cells were measured using a DCF-DA fluorescent dye. Cells were exposed to either dimethyl sulfoxide or 50 μM genistein for 24 hours. Images of cellular fluorescence were acquired by using a live cell image microscope. (C) The protein level of Nrf2 in HCC1937 with and without genistein treatment for 72 hours was measured by Western blot analysis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase .

    Journal: Journal of Cancer Prevention

    Article Title: Genistein Inhibits Proliferation of BRCA1 Mutated Breast Cancer Cells: The GPR30-Akt Axis as a Potential Target

    doi: 10.15430/JCP.2019.24.4.197

    Figure Lengend Snippet: Effects of genistein on GPR30 expression and reactive oxygen species (ROS) generation. (A) MDA-MB-231 and HCC1937 cells were treated with 50 μM Genistein. After 48 hours of incubation, the mRNA expression of GPR30 was assessed by reverse transcription-PCR. (B) The intracellular ROS levels in genistein treated MDA-MB-231 and HCC1937 cells were measured using a DCF-DA fluorescent dye. Cells were exposed to either dimethyl sulfoxide or 50 μM genistein for 24 hours. Images of cellular fluorescence were acquired by using a live cell image microscope. (C) The protein level of Nrf2 in HCC1937 with and without genistein treatment for 72 hours was measured by Western blot analysis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase .

    Article Snippet: GPR30 and Nrf2 primary antibodies were supplied from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Multiple Displacement Amplification, Incubation, Polymerase Chain Reaction, Fluorescence, Microscopy, Western Blot

    Phase II genes expression in A2780 cells after CA and DMCA treatment in the presence of catalase. (a) Nrf2 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); (b) GSTP1 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); (c) HO-1 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); n =3; data represented as mean±SD.

    Journal: Redox Biology

    Article Title: The role of the catecholic and the electrophilic moieties of caffeic acid in Nrf2/Keap1 pathway activation in ovarian carcinoma cell lines

    doi: 10.1016/j.redox.2014.11.012

    Figure Lengend Snippet: Phase II genes expression in A2780 cells after CA and DMCA treatment in the presence of catalase. (a) Nrf2 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); (b) GSTP1 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); (c) HO-1 mRNA levels after CA and DMCA 6 h treatments (black) and the same treatments in presence of 300 U of catalase in cell media (white); n =3; data represented as mean±SD.

    Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit-antihuman Nrf2 antibody (Abcam, Cambridge, Massachusetts) diluted according to manufacturer's instructions.

    Techniques: Expressing

    Effects of CA (black), DMCA (white) and HCA (gray) treatments on Nrf2 and phase II mRNA levels in A2780/cisR cells. (a) Effects of CA, DMCA and HCA treatments on Nrf2 mRNA levels; (b) effects of CA, DMCA and HCA treatments on GSTP1 mRNA levels; (c) effects of CA, DMCA and HCA treatments on GSR1 mRNA levels; (d) effects of CA, DMCA and HCA treatments on GCLM mRNA levels; (e) effects of CA, DMCA and HCA treatments on HO-1 mRNA levels; (f) basal mRNA levels of untreated cells. Data are expressed as n -fold of control (untreated cells) normalized to GAPDH ±SD of 6 independent experiments each conducted in triplicate, ⁎ p

    Journal: Redox Biology

    Article Title: The role of the catecholic and the electrophilic moieties of caffeic acid in Nrf2/Keap1 pathway activation in ovarian carcinoma cell lines

    doi: 10.1016/j.redox.2014.11.012

    Figure Lengend Snippet: Effects of CA (black), DMCA (white) and HCA (gray) treatments on Nrf2 and phase II mRNA levels in A2780/cisR cells. (a) Effects of CA, DMCA and HCA treatments on Nrf2 mRNA levels; (b) effects of CA, DMCA and HCA treatments on GSTP1 mRNA levels; (c) effects of CA, DMCA and HCA treatments on GSR1 mRNA levels; (d) effects of CA, DMCA and HCA treatments on GCLM mRNA levels; (e) effects of CA, DMCA and HCA treatments on HO-1 mRNA levels; (f) basal mRNA levels of untreated cells. Data are expressed as n -fold of control (untreated cells) normalized to GAPDH ±SD of 6 independent experiments each conducted in triplicate, ⁎ p

    Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit-antihuman Nrf2 antibody (Abcam, Cambridge, Massachusetts) diluted according to manufacturer's instructions.

    Techniques: High Content Screening

    Effect of CA on Nrf2 translocation in A2780 cells. The figure is a representative ( n = 6) result for confocal laser-scanning. (a) A2780 cells stained for Nrf2 (FITC-green) and for nuclei (Hoescht-blue). The cells were either untreated (panel A) or incubated with 50 µM CA (panel B), 1 µM trigonelline (panel C) or both (panel D) for 6 h at 37 °C in an atmosphere of 5% (v/v) CO 2 . (b) Ratio between cytoplasmic and nuclear Nrf2 measured by color analysis (Fluoview1 software, Olympus, Hamburg, Germany). p =0.003 CA vs. control. All pictures are in the same scale.

    Journal: Redox Biology

    Article Title: The role of the catecholic and the electrophilic moieties of caffeic acid in Nrf2/Keap1 pathway activation in ovarian carcinoma cell lines

    doi: 10.1016/j.redox.2014.11.012

    Figure Lengend Snippet: Effect of CA on Nrf2 translocation in A2780 cells. The figure is a representative ( n = 6) result for confocal laser-scanning. (a) A2780 cells stained for Nrf2 (FITC-green) and for nuclei (Hoescht-blue). The cells were either untreated (panel A) or incubated with 50 µM CA (panel B), 1 µM trigonelline (panel C) or both (panel D) for 6 h at 37 °C in an atmosphere of 5% (v/v) CO 2 . (b) Ratio between cytoplasmic and nuclear Nrf2 measured by color analysis (Fluoview1 software, Olympus, Hamburg, Germany). p =0.003 CA vs. control. All pictures are in the same scale.

    Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit-antihuman Nrf2 antibody (Abcam, Cambridge, Massachusetts) diluted according to manufacturer's instructions.

    Techniques: Translocation Assay, Staining, Incubation, Software

    Inhibition of Nrf2 and phase II genes induction by trigonelline in A2780 and A2780cisR cells. Cells were treated with 50 µM CA, 1 µM trig or in combination of both for 6 h and mRNA levels were measured by qRT-PCR. (a) Nrf2 mRNA levels after CA and/or trigonelline treatments for 6 h in A2780 cells (black) and in A2780cisR cells (white); (b) GSTP1 mRNA levels after CA and/or trigonelline treatments for 6 h in A2780 cells (black) and in A2780cisR cells (white); (c) HO-1 mRNA levels after CA and/or trigonelline treatments for 6 h in A2780 cells (black) and in A2780cisR cells (white). n =3; data represented as mean±SD. ⁎ p

    Journal: Redox Biology

    Article Title: The role of the catecholic and the electrophilic moieties of caffeic acid in Nrf2/Keap1 pathway activation in ovarian carcinoma cell lines

    doi: 10.1016/j.redox.2014.11.012

    Figure Lengend Snippet: Inhibition of Nrf2 and phase II genes induction by trigonelline in A2780 and A2780cisR cells. Cells were treated with 50 µM CA, 1 µM trig or in combination of both for 6 h and mRNA levels were measured by qRT-PCR. (a) Nrf2 mRNA levels after CA and/or trigonelline treatments for 6 h in A2780 cells (black) and in A2780cisR cells (white); (b) GSTP1 mRNA levels after CA and/or trigonelline treatments for 6 h in A2780 cells (black) and in A2780cisR cells (white); (c) HO-1 mRNA levels after CA and/or trigonelline treatments for 6 h in A2780 cells (black) and in A2780cisR cells (white). n =3; data represented as mean±SD. ⁎ p

    Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit-antihuman Nrf2 antibody (Abcam, Cambridge, Massachusetts) diluted according to manufacturer's instructions.

    Techniques: Inhibition, Quantitative RT-PCR

    Nrf2 and GSTP1 expression in A2780 cells after CA and DMCA treatments under normal growth condition and in the absence of oxygen. Nrf2 (black) and GSTP1 (white) mRNA levels after CA and DMCA treatments for 6 h. n =4; data represented as mean±SD. ⁎ p

    Journal: Redox Biology

    Article Title: The role of the catecholic and the electrophilic moieties of caffeic acid in Nrf2/Keap1 pathway activation in ovarian carcinoma cell lines

    doi: 10.1016/j.redox.2014.11.012

    Figure Lengend Snippet: Nrf2 and GSTP1 expression in A2780 cells after CA and DMCA treatments under normal growth condition and in the absence of oxygen. Nrf2 (black) and GSTP1 (white) mRNA levels after CA and DMCA treatments for 6 h. n =4; data represented as mean±SD. ⁎ p

    Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit-antihuman Nrf2 antibody (Abcam, Cambridge, Massachusetts) diluted according to manufacturer's instructions.

    Techniques: Expressing