Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: Information of primary antibodies.

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Concentration Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: Primer sequence list.

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Sequencing

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: AKBA can activate the expression of Nrf2 pathway to reduce oxidative stress. (A) Pictures of the fluorescent expression of Nrf2 in the spinal cord. The white scale is 20 μm. (B) Quantitative analysis of Nrf2 fluorescence pictures on day 7. (C) Nrf2/HO‐1 protein expression map. (D) Quantitative analysis of Nrf2 protein on day 7. (E) Quantitative analysis of HO‐1 protein on day 7. (F) The mRNA relative expression level of Nrf2 on the 7th day. (G) Relative mRNA expression level of HO‐1 on day 7. (H) CAT content. (I) GSH‐PX content. (J) T‐AOC content. (K) SOD enzyme activity. (L) MDA content. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Expressing, Fluorescence, Activity Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: AKBA can increase the anti‐inflammatory expression of macrophages induced by LPS and reduce the release of ROS. (A) Fluorescent expression map of CD206. The white scale is 20 μm. (B) Fluorescence quantitative analysis diagram of CD206. (C) Protein expression map of ARG‐1 and IL‐10. (D) Protein quantitative analysis chart of ARG‐1. (E) Protein quantitative analysis chart of IL‐10. (F) Fluorescence quantitative analysis of ROS. (G) CD206 flow cytometry results. (H) Expression map of ROS. The white scale is 20 μm. (I) Protein expression map of Nrf2 pathway. (J, K) Protein Quantitative Analysis of Nrf2, HO‐1. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Expressing, Fluorescence, Flow Cytometry

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: Nrf2 inhibitors attenuate the ability of AKBA to reduce the iNOS expression. (A) The fluorescence expression map of iNOS. The white scale is 20 μm. (B) Fluorescence quantitative analysis diagram of iNOS. (C) The expression level of iNOS detected by ELISA. (D) Detection level of IL‐1β detected by ELISA. (E) The detection level of TNF‐α detected by ELISA. (F) Protein expression map of TNF‐α and IL‐1β. (G, H) Protein quantitative analysis of TNF‐α, IL‐1β. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: Nrf2 inhibitors attenuate the ability of AKBA to increase the CD206 expression. (A) Fluorescent expression map of CD206. The white scale is 20 μm. (B) Fluorescence quantitative analysis of CD206. (C) Protein expression maps of ARG‐1 and IL‐10. (D, E) Protein quantitative analysis of ARG‐1, IL‐10. (F) Expression map of ROS. The white scale is 50 μm. (G) Protein expression map of Nrf2/HO‐1. (H, I) Protein quantitative analysis of Nrf2 and HO‐1. (J) Quantitative analysis of ROS. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Expressing, Fluorescence

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: Nrf2 inhibitors attenuate the ability of AKBA to promote Schwann cell migration. (A) Detection of S100 at the site of spinal cord injury. (B) AKBA‐treated macrophage culture promotes the migration ability of Schwann cells. (C) Statistical graph of Schwann cell migration. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Migration

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: AKBA promotes rat macrophage polarization and Schwann cell migration. (A) iNOS fluorescence image. Red fluorescence indicates iNOS. (B) iNOS fluorescence quantitative analysis chart. (C) CD206 fluorescence image. Red fluorescence indicates CD206. (D) CD206 fluorescence quantitative analysis chart. (E) CD206 flow chart. (F) CD206 flow cytometry analysis diagram. (G) ROS staining picture. (H) ROS fluorescence analysis chart. (I) The migration ability of rat macrophage culture medium on SC cells in each group. (J) Analysis of Schwann cell migration numbers. (K) Protein expression diagram of TNF‐α and IL‐1β. (L) Protein quantitative analysis chart of IL‐1β. (M) Quantitative analysis chart of TNF‐α. (N) Protein expression diagram of ARG‐1 and IL‐10. (O) ARG‐1 protein quantitative analysis chart. (P) Quantitative protein analysis chart of IL‐10. (Q) Protein expression diagram of Nrf2 and HO‐1. Protein quantitative analysis chart of (R) Nrf2. (S) HO‐1 protein quantitative analysis chart. C represents the blank group, C + M represents the blank plus ML385 group, C + A represents the blank plus AKBA group, and C + A + M represents the blank plus AKBA and ML385. L represents the LPS group, L + A represents the LPS + AKBA group, L + M represents the LPS + ML385 group, and L + A + M represents the LPS + AKBA + ML385 group. The lowercase letters above the statistical graph indicate differences. The same letters indicate no significant difference, and different letters indicate significant differences ( p < 0.05).

Article Snippet: It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed.

Techniques: Migration, Fluorescence, Flow Cytometry, Staining, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: Primer sequences used in this paper.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: Antibodies used in this paper.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Reverse Transcription

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: Human telomerase reverse transcriptase (hTERT) and NRF2 are highly expressed in CRC tissues and associated with poor diagnosis. (A) hTERT expression in CRC tissues and adjacent normal tissues was investigated in Oncomine database (Cohort1). (B) NRF2 expression in CRC tissues and adjacent normal tissues was investigated in Oncomine database (Cohort2). (C) hTERT mRNA expression in CRC tissues and paired adjacent normal tissues was identified by qRT-PCR (Cohort3). (D) NRF2 mRNA expression in CRC tissues and paired adjacent normal tissues was identified by qRT-PCR (Cohort3). (E) Regulation analysis of the correlation between hTERT and NRF2 (Cohort3). Each point represents one cancer sample. (F) Representative immunohistochemical staining and expression level statistics of hTERT and NRF2 in CRC tissues and paired adjacent normal tissues (Cohort4). (G) Regulation analysis of the correlation between hTERT and NRF2 (Cohort4). Each point represents one cancer sample. (H) Kaplan–Meier analysis of the overall survival of CRC patients with different hTERT expression levels ( p < 0.05, log-rank test). (I) Kaplan–Meier analysis of the overall survival of CRC patients with different NRF2 expression levels ( p < 0.05, log-rank test). (J) Kaplan–Meier analysis of the overall survival of CRC patients with different hTERT and NRF2 expression levels ( p < 0.05, log-rank test). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Reverse Transcription, Biomarker Discovery, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: Human telomerase reverse transcriptase (hTERT) upregulates NRF2 expression by promoting NRF2 transcriptional activity. (A) hTERT-knockdown cells were constructed by transfecting sh1-hTERT and sh2-hTERT lenviral respectively into HCT 116 cells. The hTERT and NRF2 mRNA expression level after downregulation of hTERT were identified by qRT-PCR. (B) hTERT and NRF2 protein expression levels after downregulation of hTERT were detected by western blotting (Left). Statistical analysis of western blotting (Right). (C) hTERT-overexpressed cell was constructed by transfecting hTERT lenviral into SW620 cells. The hTERT and NRF2 mRNA expression level after upregulation of hTERT was identified by qRT-PCR. (D) hTERT and NRF2 protein expression levels after overexpression of hTERT were detected by western blotting (Left). Statistical analysis of western blotting (Right). (E) Luciferase activity of the NRF2 promoter was detected after downregulation of hTERT. (F) Luciferase activity of NRF2 was detected after overexpression of hTERT. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Reverse Transcription, Expressing, Activity Assay, Knockdown, Construct, Quantitative RT-PCR, Western Blot, Over Expression, Luciferase

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: Human telomerase reverse transcriptase (hTERT) promotes colorectal cancer cell proliferation and metastasis by upregulating NRF2. (A) CCK8 assays were performed to detect cell proliferation after downregulation of hTERT but an increase in NRF2 expression. (B) Colony formation assays were performed after downregulation of hTERT but an increase in NRF2 expression. (C) Statistical analysis of the colony numbers. (D) Migration and invasion assays were performed after downregulation of hTERT but an increase in NRF2 expression. (E) Statistical analysis of the migration cell numbers. (F) Statistical analysis of the invasion cell numbers. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Reverse Transcription, Expressing, Migration

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: Human telomerase reverse transcriptase (hTERT) increases NRF2 expression by recruiting YBX1 to bind to the NRF2 promoter. (A) Flow chart for screening of potential hTERT-recruited NRF2 transcription factors. (B) The YBX1 and NRF2 mRNA expression level was identified by qRT-PCR after downregulation of YBX1. (C) Luciferase activity of the NRF2 promoter was detected after downregulation of YBX1. (D) NRF2 promoter with a 5′ biotin label was used to pull-down YBX1. Mock beads were used as a negative control. (E) Diagrammatic drawing of NRF2 P2 fragment. The P2 fragment was divided into 5 fragments. (F) YBX1 antibody was used to immunoprecipitate binding fragments of the NRF2 promoter under the condition of hTERT overexpression and fragments were identified by ChIP-qPCR with six primers (Left). Statistical analysis of ChIP-qPCR. (G) Luciferase activity of P2 fragment containing different mutant sites was detected after YBX1 overexpression. (H) HCT116 cell lysates were prepared for separate IP with hTERT and YBX1 antibody and then evaluated via western blotting. (I) The subcellular localization and the colocalization of hTERT and YBX1 were examined in SW620 cells via dual immunofluorescence using confocal microscopy. (J) YBX1 in cell nuclei and cytoplasm was identified by western blotting after overexpression of Flag-hTERT. (K) The NRF2 mRNA expression level was identified by qRT-PCR after overexpression of hTERT and simultaneous knockdown of YBX1. (L) Luciferase activity of the NRF2 promoter was detected after overexpression of hTERT and simultaneous knockdown of YBX1. (M) The NRF2 protein level was identified by western blotting after overexpression of hTERT and simultaneous knockdown of YBX1 (Left). Statistical analysis of western blotting (Right). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Reverse Transcription, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Negative Control, Binding Assay, Over Expression, ChIP-qPCR, Mutagenesis, Western Blot, Immunofluorescence, Confocal Microscopy, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: YBX1 is responsible for upregulation of NRF2 expression and CRC proliferation and migration. (A) The YBX1 and NRF2 mRNA expression level were identified by qRT-PCR after downregulation of YBX1 and simultaneous overexpression of NRF2. (B) The YBX1 and NRF2 protein expression level were identified via western blotting after downregulation of YBX1 and simultaneous overexpression of NRF2 (Left). Statistical analysis of western blotting (Right). (C) CCK8 assays were performed to detect cell proliferation after downregulation of YBX1 but an increase in NRF2 expression. (D) Colony formation assays were performed after downregulation of YBX1 but an increase in NRF2 expression (Left). Statistical analysis of the colony numbers (Right). (E) Migration and invasion assays were performed after downregulation of YBX1 but an increase in NRF2 expression. (F) Statistical analysis of the migration cell numbers. (G) Statistical analysis of the invasion cell numbers. (H) The YBX1 and NRF2 mRNA expression level were identified by qRT-PCR after overexpression of YBX1 and simultaneous downregulation of NRF2. (I) The YBX1 and NRF2 protein expression level were identified via western blotting after overexpression of YBX1 and simultaneous downregulation of NRF2 (Left). Statistical analysis of western blotting (Right). (J) CCK8 assays were performed to detect cell proliferation after overexpression of YBX1 and downregulation of NRF2. (K) Colony formation assays were performed after overexpression of YBX1 and downregulation of NRF2 (Left). Statistical analysis of the colony numbers (Right). (L) Migration and invasion assays were performed after overexpression of YBX1 and downregulation of NRF2. (M) Statistical analysis of the migration cell numbers. (N) Statistical analysis of the invasion cell numbers. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Expressing, Migration, Quantitative RT-PCR, Over Expression, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: YBX1 is highly expressed in CRC and associated with poor prognosis. (A) YBX1 expression in CRC tissues and adjacent normal tissues was investigated in Oncomine database. (B) YBX1 mRNA expression in CRC tissues and paired adjacent normal tissues was identified by qRT-PCR. (C) Regulation analysis of the correlation between YBX1 and NRF2. Each point represents one cancer sample. (D) Representative immunohistochemical staining and expression level statistics of YBX1 in CRC tissues and paired adjacent normal tissues. (E) Regulation analysis of the correlation between YBX1 and NRF2. Each point represents one cancer sample. (F) Kaplan–Meier analysis of the overall survival of CRC patients with different YBX1 expression levels ( p < 0.05, log-rank test). (G) Kaplan–Meier analysis of the overall survival of CRC patients with different YBX1 and NRF2 expression levels ( p < 0.05, log-rank test). * p < 0.05;** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: hTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression

doi: 10.3389/fcell.2021.658101

Figure Lengend Snippet: Model for transcriptional regulation of NRF2 by hTERT via recruitment of YBX1 in CRC proliferation and migration. In this model, hTERT recruits YBX1 to form a transcriptional complex, which binds to the NRF2 promoter to promote NRF2 expression, thus promoting CRC proliferation and migration.

Article Snippet: The 5-biotinylated DNA of the 5-flanking region of the NRF2 promoter was immobilized to streptavidin beads following the manufacturer’s protocol (Dynabeads ® kilobase BINDERTM kit, Invitrogen Dynal AS, Oslo, Norway).

Techniques: Migration, Expressing

Journal: Redox Biology

Article Title: MCL attenuates atherosclerosis by suppressing macrophage ferroptosis via targeting KEAP1/NRF2 interaction

doi: 10.1016/j.redox.2023.102987

Figure Lengend Snippet: MCL enhances NRF2 nuclear translocation in macrophages and ApoE−/− mice. In macrophages, the nuclear lysate was extracted to analysis NRF2 expression. In macrophages and aorta, Nuclear and cytoplasmic lysate were extracted to analysis NRF2 expression. (A, B). Western blot is used to evaluate NRF2 levels. N = 3; (C, D). Confocal fluorescent staining is used to evaluate the nuclear translocation of NRF2. N = 6; (E, F). The protein levels of NRF2 in the aorta nuclear lysates of ApoE −/− mice. N = 5; (G) The enrichment ability of NRF2 on GPX4 and xCT promoter using ChIP-qPCR; ***p＜0.001, vs control or ND group. ###p < 0.001, vs ox-LDL or HD group.

Article Snippet: The binding of NRF2 to GPX4 and xCT promoter was analyzed using Pierce™ Agarose ChIP Kit (Thermo Fisher).

Techniques: Translocation Assay, Expressing, Western Blot, Staining

Journal: Frontiers in Aging Neuroscience

Article Title: DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

doi: 10.3389/fnagi.2015.00244

Figure Lengend Snippet: Upregulation of mRNA and protein levels of Nrf2 by 5-Aza treatment in N2a/APPswe cells . N2a/APPswe cells were treated with 0 μM 5-Aza (control), 0.1% DMSO, 5-Aza (3 and 5 μM) for 72 h. The expressions of Nrf2 and β-actin mRNA were measured by Q-PCR using total RNA from N2a/APPswe cells. β-Actin was assessed as a loading control (A) . The protein expressions of total Nrf2 (T-Nrf2) and β-actin were measured by Western blot analysis using lysates from N2a/APPswe, normalized to β-actin expression (B) . All data were represented as a mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: Results have shown that Nrf2 promoter methylation decreased to 39 out of 50 (78%) in the first five CpG sites after treated with 5 μM 5-Aza (chi-square test, P < 0.05) (Figure B), indicating that the first five CpG sites of Nrf2 promoter have been demethylated by 5-Aza treatment.

Techniques: Control, Western Blot, Expressing

Journal: Frontiers in Aging Neuroscience

Article Title: DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

doi: 10.3389/fnagi.2015.00244

Figure Lengend Snippet: The first five CpG sites are specifically demethylated by 5-Aza treatment . N2a/APPswe cells were treated with 0.1% DMSO, 5 μM 5-Aza for 72 h. The methylation patterns of the first five CpG sites of promoter Nrf2 gene in N2a/APPswe cells were performed using bisulfite genomic sequencing (BGS) as described in Section “ .” Black circles indicate methylated CpGs, and open circles indicate unmethylated CpGs. The first five CpGs were hypermethylated in N2a/APPswe cells, which were treated with 0.1% DMSO (98% methylated) (A) and cells treated with 5 μM 5-Aza for 72 h (78% methylated) (chi-square test, P < 0.05) (B) .

Article Snippet: Results have shown that Nrf2 promoter methylation decreased to 39 out of 50 (78%) in the first five CpG sites after treated with 5 μM 5-Aza (chi-square test, P < 0.05) (Figure B), indicating that the first five CpG sites of Nrf2 promoter have been demethylated by 5-Aza treatment.

Techniques: Methylation, Genomic Sequencing

Journal: Frontiers in Aging Neuroscience

Article Title: DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

doi: 10.3389/fnagi.2015.00244

Figure Lengend Snippet: 5-Aza promotes the nuclear translocation of Nrf2 in N2a/APPswe cells . N2a/APPswe cells were treated with 0 μM 5-Aza (control), 0.1% DMSO, 5-Aza (3 and 5 μM) for 72 h. The protein expressions of N-Nrf2 (nucleus protein of Nrf2) and Lamin B were measured by Western blot using nuclear protein from N2a/APPswe cells. Lamin B was assessed as a loading control (A) . Immunofluorescence staining was used to observe the distribution of Nrf2 (B) . N2a/APPswe cells were treated with 5 μM 5-Aza for 72 h and labeled with anti-Nrf2 antibody (red) and DAPI (blue), APPswe were labeled with GFP (green). Scale bar 50 μm. All data were represented as a mean ± SD of three independent experiments. ** P < 0.01.

Article Snippet: Results have shown that Nrf2 promoter methylation decreased to 39 out of 50 (78%) in the first five CpG sites after treated with 5 μM 5-Aza (chi-square test, P < 0.05) (Figure B), indicating that the first five CpG sites of Nrf2 promoter have been demethylated by 5-Aza treatment.

Techniques: Translocation Assay, Control, Western Blot, Immunofluorescence, Staining, Labeling

Journal: Frontiers in Aging Neuroscience

Article Title: DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

doi: 10.3389/fnagi.2015.00244

Figure Lengend Snippet: 5-Aza increases mRNA and protein expression levels of Nrf2 downstream gene NQO1 . N2a/APPswe cells were treated with 0 μM 5-Aza (control), 0.1% DMSO, 5-Aza (3 and 5 μM) for 72 h. The protein expressions of NQO1 and β-actin were measured by Western blot analysis using total protein from N2a/APPswe. β-Actin was assessed as a loading control. All data were represented as a mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: Results have shown that Nrf2 promoter methylation decreased to 39 out of 50 (78%) in the first five CpG sites after treated with 5 μM 5-Aza (chi-square test, P < 0.05) (Figure B), indicating that the first five CpG sites of Nrf2 promoter have been demethylated by 5-Aza treatment.

Techniques: Expressing, Control, Western Blot

Journal: iScience

Article Title: Mitoquinone alleviates osteoarthritis progress by activating the NRF2-Parkin axis

doi: 10.1016/j.isci.2023.107647

Figure Lengend Snippet: MitoQ activated antioxidant system and mitophagy in chondrocytes Chondrocytes were exposed to IL-1β and MitoQ (0.125,0.25, and 0.5 μM) for 24 h. (A) The chondrocytes were treated with IL-1β for 1, 6, 12, 24, and 48 h, and then whole-cell lysates together with nuclear lysates were prepared for western blot. (B, C) The levels of NRF2, HO-1, and NQO1. (D) Nrf2 expression was examined by qRT-PCR. (E–G) Nuclear translocation of Nrf2 was observed by immunofluorescence (scale bar: 50 μm) (F and G) The levels of Parkin, PINK1, and LC3 II. (H) Parkin expression was examined by qRT-PCR. (I) Chondrocytes were stained with Mitotracker (mitochondria marker, red) and Parkin (green) (scale bar: 25 μm). ∗p < 0.05 vs. the control group, ∗∗p < 0.01 vs. the control group, #p < 0.05 vs. the IL-1β treated group, ##p < 0.01 vs. the IL-1β treated group, Data are represented as mean ± SD.

Article Snippet: The immunofluorescence results revealed that NRF2 was found largely in the cytoplasm of normal chondrocytes, while green-stained NRF2 was observed in the chondrocyte nucleus in the IL-1β group and MitoQ further promoted the nuclear localization of NRF2( E).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Translocation Assay, Immunofluorescence, Staining, Marker, Control

Journal: iScience

Article Title: Mitoquinone alleviates osteoarthritis progress by activating the NRF2-Parkin axis

doi: 10.1016/j.isci.2023.107647

Figure Lengend Snippet: NRF2 antioxidant pathway mediated the protection of MitoQ on chondrocytes After silencing NRF2, chondrocytes were exposed to IL-1β and MitoQ for 24 h. (A and B) The chondrocytes whole-cell lysates together with nuclear lysates were prepared for western blot. The levels of NRF2, HO-1, and NQO1. (C and D) The levels of iNOS, COX2, MMP13, and MMP3. (E and F) The GPX4 expression. (G and H) The levels of Parkin and PINK1. ∗p < 0.05 vs. the IL-1β + negative control group, ∗∗p < 0.01 vs. the IL-1β + negative control group, #p < 0.05 vs. the IL-1β + MitoQ + negative control group, ##p < 0.01 vs. the IL-1β + MitoQ + negative control group, Data are represented as mean ± SD.

Article Snippet: The immunofluorescence results revealed that NRF2 was found largely in the cytoplasm of normal chondrocytes, while green-stained NRF2 was observed in the chondrocyte nucleus in the IL-1β group and MitoQ further promoted the nuclear localization of NRF2( E).

Techniques: Western Blot, Expressing, Negative Control

Journal: iScience

Article Title: Mitoquinone alleviates osteoarthritis progress by activating the NRF2-Parkin axis

doi: 10.1016/j.isci.2023.107647

Figure Lengend Snippet: Parkin regulated the expression of the NRF2 antioxidant pathway through negative feedback After Parkin knockdown, chondrocytes were exposed to IL-1β and MitoQ for 24 h. (A) The level of Parkin. (B and C) The levels of Parkin and PINK1. (D and E) The protein levels of iNOS, MMP3, and COL2A1. (F) The mRNA level of Col2a1. (G and H) The GPX4 level was measured by western blot and qRT-PCR. (I and J) The protein levels of NRF2, HO-1, and NQO1, and the mRNA level of Nrf2. (K and L) The levels of NRF2, HO-1, and NQO1 treated by MitoQ with negative control or si-Parkin. (M and N) The expression of iNOS, MMP13, and COL2A1. ∗p < 0.05 vs. the IL-1β + negative control group, ∗∗p < 0.01 vs. the IL-1β + negative control group, #p < 0.05 vs. the IL-1β + negative control or IL-1β + MitoQ + negative control group, ##p < 0.01 vs. the IL-1β + negative control or IL-1β + MitoQ + negative control group, Data are represented as mean ± SD.

Article Snippet: The immunofluorescence results revealed that NRF2 was found largely in the cytoplasm of normal chondrocytes, while green-stained NRF2 was observed in the chondrocyte nucleus in the IL-1β group and MitoQ further promoted the nuclear localization of NRF2( E).

Techniques: Expressing, Knockdown, Western Blot, Quantitative RT-PCR, Negative Control

Journal: iScience

Article Title: Mitoquinone alleviates osteoarthritis progress by activating the NRF2-Parkin axis

doi: 10.1016/j.isci.2023.107647

Figure Lengend Snippet: MitoQ attenuated DMM-induced cartilage degeneration After DMM surgery, MitoQ (0.1 mg/kg and 1 mg/kg) or vehicle was intra-articularly injected into mice’ knee joints twice a week for 8 weeks. (A and B) Safranin O staining/fast green was used to measure cartilage degeneration. The OARSI scores were used to assess the progression of OA. Scale bar: 200 μm and 100 μm (The sham and the DMM +0.1 mg/kg MitoQ group (n = 8), the DMM and the DMM +1 mg/kg MitoQ group (n = 9)). (C–H) Immunohistochemistry staining was used to measure COL2A1, NRF2, HO-1, GPX4, Parkin, and PINK1 levels. Quantification of COL2A1-, MMP13-, NRF2- HO-1-, GPX4-, Parkin-, and PINK1-positive staining cells in vivo (scale bar: 100 μm). ∗p < 0.05 vs. the sham group, #p < 0.05 vs. the DMM group, ##p < 0.01 vs. the DMM group, n = 7 or 8. Data are represented as mean ± SD.

Article Snippet: The immunofluorescence results revealed that NRF2 was found largely in the cytoplasm of normal chondrocytes, while green-stained NRF2 was observed in the chondrocyte nucleus in the IL-1β group and MitoQ further promoted the nuclear localization of NRF2( E).

Techniques: Injection, Staining, Immunohistochemistry, In Vivo

Journal: iScience

Article Title: Mitoquinone alleviates osteoarthritis progress by activating the NRF2-Parkin axis

doi: 10.1016/j.isci.2023.107647

Figure Lengend Snippet: Possible regulatory mechanism of MitoQ’s anti-OA function MitoQ relieved cartilage degeneration by activating the antioxidant system and mitophagy and inhibiting ferroptosis via the NRF2/Parkin axis. Parkin regulated the expression of the NRF2 antioxidant pathway through negative feedback.

Article Snippet: The immunofluorescence results revealed that NRF2 was found largely in the cytoplasm of normal chondrocytes, while green-stained NRF2 was observed in the chondrocyte nucleus in the IL-1β group and MitoQ further promoted the nuclear localization of NRF2( E).

Techniques: Expressing

Journal: iScience

Article Title: Mitoquinone alleviates osteoarthritis progress by activating the NRF2-Parkin axis

doi: 10.1016/j.isci.2023.107647

Figure Lengend Snippet:

Article Snippet: The immunofluorescence results revealed that NRF2 was found largely in the cytoplasm of normal chondrocytes, while green-stained NRF2 was observed in the chondrocyte nucleus in the IL-1β group and MitoQ further promoted the nuclear localization of NRF2( E).

Techniques: Recombinant, Isolation, cDNA Synthesis, ROS Assay, Protein Extraction, Lysis, CCK-8 Assay, Staining, Software

Journal: iScience

Article Title: Mitoquinone alleviates osteoarthritis progress by activating the NRF2-Parkin axis

doi: 10.1016/j.isci.2023.107647

Figure Lengend Snippet: Primer sequence used in the RT-qPCR experiment

Article Snippet: The immunofluorescence results revealed that NRF2 was found largely in the cytoplasm of normal chondrocytes, while green-stained NRF2 was observed in the chondrocyte nucleus in the IL-1β group and MitoQ further promoted the nuclear localization of NRF2( E).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A-B. CCK-8 and EdU assays were used to detect proliferation in HEI-OC1 cells treated with LPS. As detected by CCK-8 and EdU assays, cell proliferation was markedly suppressed by LPS treatment compared to the control group. Scale bar = 100 µm in B. C. Apoptosis in HEI-OC1 cells treated with LPS was assessed via flow cytometry analysis. The apoptotic rate was increased significantly by LPS treatment. D. Western blot analysis of BCL-2 and BAX expressions in HEI-OC1 cells treated with LPS. The BAX protein level increased while the BCL-2 protein level decreased after LPS treatment. E. Western blot analysis of HDAC2 protein expression in HEI-OC1 cells treated with LPS, DEX, or DEX+TA. HDAC2 protein levels were decreased in HEI-OC1 cells treated with LPS alone, while its levels were partly reversed by DEX treatment and totally reversed by treatment with both DEX and TA (**P < 0.01).

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: CCK-8 Assay, Control, Flow Cytometry, Western Blot, Expressing

Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A. q-PCR and western blot analyses of NRF2 and HDAC2 expressions in HEI-OC1 cells treated with LPS, DEX or DEX+TA. TA increased NRF2 and HDAC2 mRNA and protein levels in DEX-treated HEI-OC1 cells (**P < 0.01). B. RT-qPCR was used to check gene over-expression of NRF2 in HEI-OC1 cells. NRF2 was successfully over-expressed in HEI-OC1 cells. C. HDAC2 mRNA level was detected via RT-qPCR in HEI-OC1 cells over-expressed NRF2. We detected an increased HDAC2 mRNA level in HEI-OC1 cells over-expressed NRF2 (**P < 0.01). D. Potential NRF2-binding site in the sequence of HDAC2 promoter was predicted by JASPAR database. Based on JASPAR database ( https://jaspar.genereg.net/ ) analysis, we obtained putative NRF2-binding sites in the sequence of HDAC2 promoter. The NRF2-binding site with the highest prediction score is located at 419–429 bases (ATGACACTCCA) in HDAC2 promoter sequence. E. ChIP detected enrichment of HDAC2 promoter in immunoprecipitate with Anti-NRF2. ChIP assay presented an abundant enrichment of HDAC2 promoter in immunoprecipitates with Anti-NRF2, verifying the binding between NRF2 and HDAC2 promoter. F. Luciferase activity of different reporter constructs in NRF2-overexpressing 293T cells was detected. G. DNA pull-down assay followed by western blot was used to detect NRF2 expression in the complex pulled down by Bio-HDAC2 promoter-WT. NRF2 over-expression increased the luciferase activity of HDAC2 promoter-WT while not changing the HDAC2 promoter-MUT activity (mutated sequence: TACTGTGAGGT at 419–429 bases). **P < 0.01.

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: Western Blot, Quantitative RT-PCR, Over Expression, Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Pull Down Assay, Expressing

Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A. RT-qPCR and western blot were used to measure FOXP3 expression in LPS-treated HEI-OC1 cells. FOXP3 mRNA and protein levels were increased in response to additional TA treatment (P < 0.01). B. Gene over-expression efficiency of FOXP3 in HEI-OC1 cells was tested via RT-qPCR. FOXP3 was over-expressed in HEI-OC1 cells. C. GR expression in FOXP3-over-expressing HEI-OC1 cells was measured via western blot. GR protein level was also increased in FOXP3-over-expressing HEI-OC1 cells. D. NRF2 expression in FOXP3-over-expressing HEI-OC1 cells was measured via RT-qPCR. Elevated NRF2 mRNA level was observed in FOXP3-over-expressed HEI-OC1 cells. E. Potential FOXP3-binding sites in NRF2 promoter sequence were predicted by JASPAR database. According to JASPAR database analysis, the predicted FOXP3-binding site with the highest prediction score is located at 864–870 bases (GCAAACA) in NRF2 promoter sequence. F. Luciferase activity of different reporter constructs in FOXP3-over-expressing 293T cells was detected by luciferase reporter assay. The luciferase activity of pGL3-NRF2 promoter-WT was enhanced by up-regulation of FOXP3 in 293T cells, while that of pGL3-NRF2 promoter-MUT (mutated sequence: CGTTTGT at 864–870 bases) was not affected. G. ChIP assay was performed to detect the physical binding between FOXP3 and NRF2 promoter. ChIP assay uncovered that NRF2 promoter was highly enriched in Anti-FOXP3-precipitated complex. H. DNA pull-down assay followed by western blot was used to detect FOXP3 expression in the complex pulled down by Bio-NRF2 promoter-WT. DNA pull-down assay validated the effectiveness of the binding sites between FOXP3 and NRF2 promoter. I. The knock-down efficiency of si-NRF2 plasmids was checked via RT-qPCR. All three siRNAs had high efficacy of NRF2 knock-down. J. HDAC2 expression was measured via RT-qPCR and western blot in HEI-OC1 cells. The increased mRNA and protein levels of HDAC2 caused by FOXP3 over-expression were reversed by depletion of NRF2. K. FOXP3 expression was knocked down in DEX-treated HEI OC1 cells. The mRNA level of NRF2 was measured in DEX-treated HEI OC1 cells after FOXP3 knock-down by RT-qPCR. L. Protein levels of FOXP3 and NRF2 were measured in DEX-treated HEI OC1 cells after FOXP3 knock-down. The mRNA and protein levels of NRF2 increased by TA treatment while the levels were re-decreased by silencing of FOXP3 (**P < 0.01).

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Reporter Assay, Pull Down Assay, Knockdown

Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A. Interference efficiency of si-HDAC2 plasmids in LPS-induced HEI-OC1 cells was checked via RT-qPCR and western blot. si-HDAC2-1 plasmid was used in the subsequent experiments for its highest interference efficiency. B-C. CCK-8 and EdU assays were performed to assess cell proliferation. Scale bar: 100 µm. HDAC2 knock-down abrogated the promoting effect of FOXP3 over-expression on cell proliferation. D-E. Western blot of BAX and BCL-2 expressions and flow cytometry analysis were performed to evaluate apoptosis. Elevated BCL-2 protein levels and declined BAX protein levels resulting from FOXP3 up-regulation were recovered by HDAC2 deficiency. Moreover, the apoptosis rate reduced by FOXP3 over-expression was restored by HDAC2 knock-down (**P < 0.01).

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: Quantitative RT-PCR, Western Blot, Plasmid Preparation, CCK-8 Assay, Knockdown, Over Expression, Flow Cytometry

Journal: Antioxidants

Article Title: Medicarpin Increases Antioxidant Genes by Inducing NRF2 Transcriptional Level in HeLa Cells

doi: 10.3390/antiox11020421

Figure Lengend Snippet: Primer sets for real-time PCR.

Article Snippet: Human NRF2 promoter-luciferase reporter plasmid was constructed by inserting the amplified ~2 Kb sized NRF2 promoter into the modified pGL4.10-Basic vector (Promega, Madison, WI, USA), which contains AscI and PacI restriction enzyme sites in the multicloning sites.

Techniques:

Journal: Antioxidants

Article Title: Medicarpin Increases Antioxidant Genes by Inducing NRF2 Transcriptional Level in HeLa Cells

doi: 10.3390/antiox11020421

Figure Lengend Snippet: Medicarpin increases nuclear NRF2 accumulation, which results in HO-1 induction in HeLa cells. ( A ). Western blotting data shows nuclear NRF2 level after treatment with different concentrations of medicarpin for 6 h. The densitometrical analysis of nuclear NRF2 is shown in the right panel. ( B ). Western blot analysis using whole cell lysates shows the HO-1 level after treatment with the indicated concentration of medicarpin for 24 h. The densitometrical analysis of HO-1 is shown in the right panel. GAPDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. CE, cytoplasmic extract; NE, nuclear extract. The experiments were repeated three times with similar results. ** p < 0.001; *** p < 0.0001.

Article Snippet: Human NRF2 promoter-luciferase reporter plasmid was constructed by inserting the amplified ~2 Kb sized NRF2 promoter into the modified pGL4.10-Basic vector (Promega, Madison, WI, USA), which contains AscI and PacI restriction enzyme sites in the multicloning sites.

Techniques: Western Blot, Concentration Assay

Journal: Antioxidants

Article Title: Medicarpin Increases Antioxidant Genes by Inducing NRF2 Transcriptional Level in HeLa Cells

doi: 10.3390/antiox11020421

Figure Lengend Snippet: Medicarpin induces the transcriptional level of NRF2 and NRF2 target genes in HeLa cells. A. Real-time PCR analysis showed the mRNA levels of NRF2, HO-1, GCLC, and NQO-1 after treatment with different concentrations of medicarpin for 24 h in HeLa cells. Experiments were performed in triplicate. * p < 0.05; ** p < 0.001; *** p < 0.0001; NS, not significant.

Article Snippet: Human NRF2 promoter-luciferase reporter plasmid was constructed by inserting the amplified ~2 Kb sized NRF2 promoter into the modified pGL4.10-Basic vector (Promega, Madison, WI, USA), which contains AscI and PacI restriction enzyme sites in the multicloning sites.

Techniques: Real-time Polymerase Chain Reaction

Journal: Antioxidants

Article Title: Medicarpin Increases Antioxidant Genes by Inducing NRF2 Transcriptional Level in HeLa Cells

doi: 10.3390/antiox11020421

Figure Lengend Snippet: Medicarpin induces the NRF2 transcriptional level in Hela cells. Cells were treated with 50 μM of medicarpin for 6 h in the condition of transfection with an NRF2-promoter luciferase construct. NRF2-luciferase activity was measured using a Dual-Luciferase Reporter Assay kit (Promega) according to the manufacturer’s instructions. Experiments were performed in quintuplicate. *** p < 0.0001.

Article Snippet: Human NRF2 promoter-luciferase reporter plasmid was constructed by inserting the amplified ~2 Kb sized NRF2 promoter into the modified pGL4.10-Basic vector (Promega, Madison, WI, USA), which contains AscI and PacI restriction enzyme sites in the multicloning sites.

Techniques: Transfection, Luciferase, Construct, Activity Assay, Reporter Assay

Journal: Antioxidants

Article Title: Medicarpin Increases Antioxidant Genes by Inducing NRF2 Transcriptional Level in HeLa Cells

doi: 10.3390/antiox11020421

Figure Lengend Snippet: Medicarpin increases the NRF2 stability by inhibiting ubiquitin-mediated proteolysis in HeLa cells. ( A ). Cells were treated with 50 μM of medicarpin for 6 h after co-transfection with pEGFP-NRF2 and pcDNA3.1-His ubiquitin plasmids. Next, cells were lysed with RIPA and His-ubiquitinated proteins were purified using Ni-NTA agarose beads. After washing with RIPA, ubiquitinylated eGFP-NRF2 was visualized by Western blot analysis. ( B ). The relative fold change of ubiquitinylated eGFP-NRF2 was measured using a densitometer from ( A ). Experiments were performed in duplicate.

Article Snippet: Human NRF2 promoter-luciferase reporter plasmid was constructed by inserting the amplified ~2 Kb sized NRF2 promoter into the modified pGL4.10-Basic vector (Promega, Madison, WI, USA), which contains AscI and PacI restriction enzyme sites in the multicloning sites.

Techniques: Cotransfection, Purification, Western Blot