si rna targeting nrf2  (MedChemExpress)

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: GBD activates Nrf2 signaling in response to LTA. MH-S cells were pretreated with GBD for 30 min and then stimulated with LTA. (A) Confocal images indicating Nrf2 nuclear translocation 3 h poststimulation. Scale bar, 50 µm. (B) Time-course of Nrf2 mRNA expression after LTA stimulation (0, 2, 4, 6, and 8 h). (C) Nrf2 mRNA expression was determined through reverse transcription-quantitative PCR 2 h poststimulation, as described in the materials and methods section. (D) Western blot analysis of Nrf2 protein levels 3 h after LTA stimulation. Data are presented as means±SD. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 h or DMSO; ## P<0.01 and ### P<0.001 vs. DMSO+LTA. GBD glabridin; Nrf2, nuclear factor erythroid 2-related factor 2; LTA, lipoteichoic acid; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Translocation Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: GBD increases HO-1 expression through Nrf2 signaling under LTA challenge. MH-S cells were pretreated with GBD for 30 min and then stimulated with LTA. (A) Confocal images of HO-1 expression 6 h poststimulation. Scale bar, 50 µm. (B) Time-course of HO-1 mRNA expression after LTA stimulation (0, 2, 4, 6, and 8 h). (C) HO-1 mRNA expression was determined by reverse transcription-quantitative PCR. (D) HO-1 protein expression analyzed through western blotting 6 h poststimulation. Data are presented as means±SD (n=4). *P<0.05 and ***P<0.001 vs. 0 h or DMSO; ### P<0.001 vs. DMSO+LTA. GBD glabridin; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; LTA, lipoteichoic acid; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: ML385 inhibits GBD-induced Nrf2 activation in LTA-stimulated MH-S cells. MH-S cells were pretreated with DMSO (0.1%), GBD (20 µM), or ML385 (5 µM) for 30 min and then stimulated with LTA. (A) Confocal microscopy of Nrf2 nuclear localization 3 h poststimulation. Scale bar, 10 µm. (B) Quantification of Nrf2 nuclear accumulation based on nuclear mean fluorescence intensity. (C) Nrf2 mRNA expression 2 h poststimulation, analyzed through reverse transcription-quantitative PCR. Data are presented as means ± SD. ***P<0.001 vs. DMSO+LTA; ## P<0.01 vs. GBD+LTA; †† P<0.01 and ††† P<0.001 vs. ML385+LTA. GBD glabridin; Nrf2, nuclear factor erythroid 2-related factor 2; LTA, lipoteichoic acid; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Activation Assay, Confocal Microscopy, Fluorescence, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: Pharmacological and genetic inhibition of Nrf2 attenuates GBD-induced HO-1 expression. MH-S cells were pretreated with the indicated compounds for 30 min and then stimulated with LTA. (A) Immunofluorescence analysis of HO-1 expression 6 h poststimulation. Scale bar, 10 µm. (B) Immunofluorescence image evaluation of HO-1 mean fluorescence intensity. (C) HO-1 mRNA expression 6 h poststimulation, determined through RT-qPCR. (D and E) Effect of Nrf2 knockdown on GBD-induced HO-1 expression. MH-S cells were transfected with control siRNA (siNC) or Nrf2 siRNA (siNrf2) for 6 h, followed by the indicated treatments. HO-1 mRNA levels were measured by RT-qPCR. Data are presented as means ± SD. ***P<0.001 vs. DMSO+LTA (B-C) or si-Ctrl+DMSO (D and E); ## P<0.01 and ### P<0.001 vs. GBD+LTA (B-C) or si-Ctrl+DMSO+LTA (E); †† P<0.01 and ††† P<0.001 vs. ML385+LTA (B-C) or si-Ctrl+GBD+LTA (E). Nrf2, nuclear factor erythroid 2-related factor 2; GBD, glabridin; LTA, lipoteichoic acid; HO-1, heme oxygenase-1; RT-qPCR, reverse transcription-quantitative PCR; si, short interfering; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Inhibition, Expressing, Immunofluorescence, Fluorescence, Quantitative RT-PCR, Knockdown, Transfection, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: GBD inhibits LTA-induced macrophage migration through Nrf2 activation. MH-S cells were pretreated with the indicated compounds for 30 min and then stimulated with LTA. (A) Representative wound healing images 0, 6, and 24 h after LTA stimulation. (B) Quantification of wound closure. (C) Representative images of Transwell migration at 6 h and 24 h after LTA stimulation. Migrated cells on the lower surface of the membrane were fixed, stained with crystal violet and images captured under an inverted light microscope. Scale bar, 200 µm. (D) Quantification of migrated cells. Data are presented as means ± SD. ***P<0.001 vs. DMSO; ### P<0.001 vs. DMSO+LTA; † P<0.05, †† P<0.01 and ††† P<0.001 vs. GBD+LTA; ‡‡ P<0.01 vs. ML385+LTA. GBD, glabridin; LTA, lipoteichoic acid; Nrf2, nuclear factor erythroid 2-related factor 2; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Migration, Activation Assay, Membrane, Staining, Light Microscopy

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: Schematic of GBD-induced modulation of macrophage migration in vitro . Upon LTA stimulation, MH-S cells increase ROS production, activating Nrf2 and upregulating HO-1. GBD thus modulateS cell migration by increasing Nrf2 nuclear translocation and HO-1 expression. GBD, glabridin; LTA, lipoteichoic acid; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Migration, In Vitro, Translocation Assay, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Yellow Wine Polyphenolic Compounds protect against myocardial ischemia-reperfusion injury in rats by activating Nrf2 nuclear translocation to regulate the balance of mitochondrial fission and fusion

doi: 10.3389/fcvm.2025.1506388

Figure Lengend Snippet: YWPC facilitates Nrf2 nuclear translocation and activation of downstream antioxidant molecule expression. (A) Representative images of Nrf2 distribution in H9C2 cells cultured in vitro . (B,C) Western blot analysis of Keap1, Nrf2, NQO1, HO1, SOD2, GPX3 and Catalase in H9C2 cultured in vitro . ns, Not statistically significant. Data presented as mean ± SD. One-way ANOVA was used for comparisons among five groups, followed by Tukey's post hoc analysis.

Article Snippet: To further investigate the role of Nrf2 in vitro , targeted Nrf2 small interfering RNA (si-Nrf2) (Obio Technology Corp., Ltd, Shanghai, China) was used to interfere with Nrf2 expression, and Western blot analysis confirmed the effective reduction of Nrf2 synthesis by siRNA ( ).

Techniques: Translocation Assay, Activation Assay, Expressing, Cell Culture, In Vitro, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Yellow Wine Polyphenolic Compounds protect against myocardial ischemia-reperfusion injury in rats by activating Nrf2 nuclear translocation to regulate the balance of mitochondrial fission and fusion

doi: 10.3389/fcvm.2025.1506388

Figure Lengend Snippet: YWPC preconditioning improves ischemia-reperfusion-induced cardiac dysfunction via Nrf2 mediation. (A) Schematic diagram of in vivo experiments in this section. (B,C) TTC staining shows the infarct size in different groups and the corresponding quantitative data. The images display different transverse sections of the same heart from each group. (D) M-mode echocardiography. (E–H) M-mode echocardiography measurements of LVEF, LVFS, LVIDd, and LVIDs. (I) Serum cardiac injury markers (LDH, CK-MB, and cTnI) measured by ELISA. (J) Serum oxidative stress marker (MDA) measured by ELISA. YWPC, Yellow Wine Polyphenolic Compounds; TTC, 2,3,5-Triphenyltetrazolium chloride; LVEF, Left ventricular ejection fraction; LVFS, Left ventricular fractional shortening; LVIDd, Left ventricular internal diameter at end-diastole; LVIDs, left ventricular internal diameter at end-systole; LDH, lactate dehydrogenase; CK-MB, creatine kinase-MB; cTnI, cardiac troponin I; MDA, malondialdehyde; ns, Not statistically significant. Data presented as mean ± SD. Two-way ANOVA was used for comparisons among four groups, followed by Tukey's post hoc analysis. ns, Not statistically significant.

Article Snippet: To further investigate the role of Nrf2 in vitro , targeted Nrf2 small interfering RNA (si-Nrf2) (Obio Technology Corp., Ltd, Shanghai, China) was used to interfere with Nrf2 expression, and Western blot analysis confirmed the effective reduction of Nrf2 synthesis by siRNA ( ).

Techniques: In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Marker

Journal: Frontiers in Cardiovascular Medicine

Article Title: Yellow Wine Polyphenolic Compounds protect against myocardial ischemia-reperfusion injury in rats by activating Nrf2 nuclear translocation to regulate the balance of mitochondrial fission and fusion

doi: 10.3389/fcvm.2025.1506388

Figure Lengend Snippet: YWPC preconditioning improves ischemia-reperfusion-induced myocardial edema and apoptosis via Nrf2 mediation. (A) HE staining used to detect myocardial morphology. (B,C) TUNEL assay to assess myocardial apoptosis. (D) Western blot analysis of the relative protein expression of BAX, BCL-2, BAD, Cleaved Caspase3 and Caspase3 in myocardial tissue. ns, Not statistically significant. Data presented as mean ± SD. Two-way ANOVA was used for comparisons among four groups, followed by Tukey's post hoc analysis. ns, Not statistically significant.

Article Snippet: To further investigate the role of Nrf2 in vitro , targeted Nrf2 small interfering RNA (si-Nrf2) (Obio Technology Corp., Ltd, Shanghai, China) was used to interfere with Nrf2 expression, and Western blot analysis confirmed the effective reduction of Nrf2 synthesis by siRNA ( ).

Techniques: Staining, TUNEL Assay, Western Blot, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Yellow Wine Polyphenolic Compounds protect against myocardial ischemia-reperfusion injury in rats by activating Nrf2 nuclear translocation to regulate the balance of mitochondrial fission and fusion

doi: 10.3389/fcvm.2025.1506388

Figure Lengend Snippet: YWPC preconditioning regulates mitochondrial fission-fusion balance via Nrf2 mediation. (A) Representative mitochondrial images obtained by transmission electron microscopy, with representative mitochondria in the bottom images (partial enlargement of the top images) marked with light green pseudocolor to highlight mitochondrial changes. (B) Quantitative data of average mitochondrial size and the number of mitochondria per μm 2 . (C) Quantitative assessment of mitochondrial respiratory chain complex activity. (D) Representative immunoblotting images of mitochondrial OXPHOS proteins in mouse heart lysates (antibodies against Ndufb8, Sdhb, Uqcrc2, Mtco1, and Atp5a1 used as representatives for mitochondrial complex I, II, III, IV, and V). (E) Western blot analysis of the relative protein expression of p-DRP1, DRP1, MFN1, MFN2, and OPA1 in vivo . OXPHOS, Oxidative phosphorylation; ns, Not statistically significant. Data presented as mean ± SD. Two-way ANOVA was used for comparisons among four groups, followed by Tukey's post hoc analysis. ns, Not statistically significant.

Article Snippet: To further investigate the role of Nrf2 in vitro , targeted Nrf2 small interfering RNA (si-Nrf2) (Obio Technology Corp., Ltd, Shanghai, China) was used to interfere with Nrf2 expression, and Western blot analysis confirmed the effective reduction of Nrf2 synthesis by siRNA ( ).

Techniques: Transmission Assay, Electron Microscopy, Activity Assay, Western Blot, Expressing, In Vivo, Phospho-proteomics

Journal: Frontiers in Cardiovascular Medicine

Article Title: Yellow Wine Polyphenolic Compounds protect against myocardial ischemia-reperfusion injury in rats by activating Nrf2 nuclear translocation to regulate the balance of mitochondrial fission and fusion

doi: 10.3389/fcvm.2025.1506388

Figure Lengend Snippet: In vitro , YWPC reduces apoptosis in cardiomyocytes, mediated by Nrf2. (A) TUNEL staining detects cardiomyocyte apoptosis. (B) Western blot analysis of the relative protein expression of BAX, BCL-2, BAD, Cleaved Caspase3 and Caspase3 in H9C2 cells. Data presented as mean ± SD. One-way ANOVA was used for comparisons among five groups, followed by Tukey's post hoc analysis.

Article Snippet: To further investigate the role of Nrf2 in vitro , targeted Nrf2 small interfering RNA (si-Nrf2) (Obio Technology Corp., Ltd, Shanghai, China) was used to interfere with Nrf2 expression, and Western blot analysis confirmed the effective reduction of Nrf2 synthesis by siRNA ( ).

Techniques: In Vitro, TUNEL Assay, Staining, Western Blot, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Yellow Wine Polyphenolic Compounds protect against myocardial ischemia-reperfusion injury in rats by activating Nrf2 nuclear translocation to regulate the balance of mitochondrial fission and fusion

doi: 10.3389/fcvm.2025.1506388

Figure Lengend Snippet: In vitro , YWPC regulates mitochondrial fission-fusion balance in H9C2 cells, mediated by Nrf2. (A) Morphological images of mitochondria in H9C2 cells under different intervention conditions. (B,C) Western blot analysis of the relative protein expression of p-DRP1-S616, DRP1, MFN1, MFN2, and OPA1 in H9C2 cells cultured in vitro . ns, not significant. Data presented as mean ± SD. One-way ANOVA was used for comparisons among five groups, followed by Tukey's post hoc analysis. ns, Not statistically significant.

Article Snippet: To further investigate the role of Nrf2 in vitro , targeted Nrf2 small interfering RNA (si-Nrf2) (Obio Technology Corp., Ltd, Shanghai, China) was used to interfere with Nrf2 expression, and Western blot analysis confirmed the effective reduction of Nrf2 synthesis by siRNA ( ).

Techniques: In Vitro, Western Blot, Expressing, Cell Culture

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoids in lipopolysaccharide-treated HEI-OC1 cells through modulation of the FOXP3/Nrf2 signaling pathway

doi: 10.3724/abbs.2024194

Figure Lengend Snippet: TA enhances HDAC2 expression through NRF2-mediated transcription activation (A) RT-qPCR and western blot analysis 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. (B) RT-qPCR was used to assess the gene overexpression of NRF2 in HEI-OC1 cells. NRF2 was successfully overexpressed in HEI-OC1 cells. (C) HDAC2 mRNA levels were detected by RT-qPCR in HEI-OC1 cells overexpressing NRF2. We detected increased HDAC2 mRNA level in HEI-OC1 cells overexpressing NRF2. (D) A potential NRF2-binding site in the sequence of the HDAC2 promoter was predicted using the JASPAR database. The NRF2-binding site with the highest prediction score is located at bases 419–429 (ATGACACTCCA) in the HDAC2 promoter sequence. (E) ChIP detected enrichment of the HDAC2 promoter in immunoprecipitates immunoprecipitated with anti-NRF2. ChIP assay revealed abundant enrichment of the HDAC2 promoter in the immunoprecipitates with anti-NRF2, verifying the binding between NRF2 and the 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 analysis was used to detect NRF2 expression in the complex pulled down by the Bio-HDAC2 promoter-WT. NRF2 overexpression increased the luciferase activity of the HDAC2 promoter-WT but did not affect the activity of the HDAC2 promoter-MUT (mutated sequence: TACTGTGAGGT at bases 419–429). *P < 0.05 and **P < 0.01.

Article Snippet: Three small interfering RNAs (siRNAs) targeting NRF2 (si-NRF2-1, si-NRF2-2, and si-NRF2-3) or HDAC2 (si-HDAC2-1, si-HDAC2-2, and si-HDAC2-3) were synthesized by GenePharma (Shanghai, China) for the silencing of each target, and their sequences are shown in .

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

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoids in lipopolysaccharide-treated HEI-OC1 cells through modulation of the FOXP3/Nrf2 signaling pathway

doi: 10.3724/abbs.2024194

Figure Lengend Snippet: TA upregulates FOXP3 expression to activate NRF2 transcription (A) RT-qPCR and western blot analysis 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. (B) The gene overexpression efficiency of FOXP3 in HEI-OC1 cells was tested via RT-qPCR. FOXP3 was overexpressed in HEI-OC1 cells. (C) GR expression in FOXP3-overexpressing HEI-OC1 cells was measured via western blot analysis. GR protein level was also increased in FOXP3-overexpressing HEI-OC1 cells. (D) NRF2 expression in FOXP3-overexpressing HEI-OC1 cells was measured by RT-qPCR. Elevated NRF2 mRNA levels were observed in FOXP3-overexpressing HEI-OC1 cells. (E) Potential FOXP3-binding sites in the NRF2 promoter sequence were predicted using the JASPAR database. According to the JASPAR database analysis, the predicted FOXP3-binding site with the highest prediction score is located at 864–870 bases (GCAAACA) in the NRF2 promoter sequence. (F) Luciferase activity of different reporter constructs in FOXP3-overexpressing 293T cells was detected by luciferase reporter assay. The luciferase activity of pGL3-NRF2 promoter-WT was increased by the upregulation of FOXP3 in 293T cells, whereas that of pGL3-NRF2 promoter-MUT (with a mutated sequence of CGTTTGT at amino acids 864–870) was not affected. (G) ChIP assay was performed to detect the physical binding between FOXP3 and the NRF2 promoter. ChIP assay revealed that the NRF2 promoter was highly enriched in the anti-FOXP3-precipitated complex. (H) A DNA pull-down assay followed by western blot analysis was used to detect FOXP3 expression in the complex pulled down by the Bio-NRF2 promoter-WT. DNA pull-down assay validated the effectiveness of the binding sites between FOXP3 and the NRF2 promoter. (I) The knockdown efficiency of the si-NRF2 plasmid was determined by RT-qPCR. All three siRNAs had high efficacy in terms of NRF2 knockdown. (J) HDAC2 expression in HEI-OC1 cells was measured by RT-qPCR and western blot analysis. The increased mRNA and protein levels of HDAC2 caused by FOXP3 overexpression were reversed by depletion of NRF2. (K) FOXP3 expression was downregulated in DEX-treated HEI OC1 cells. The NRF2 mRNA level was measured by RT-qPCR in DEX-treated HEI OC1 cells after FOXP3 knockdown. (L) Protein levels of FOXP3 and NRF2 were measured in DEX-treated HEI OC1 cells after FOXP3 knockdown. The NRF2 mRNA and protein levels were increased by TA treatment, whereas the levels were decreased by silencing of FOXP3. *P < 0.05 and **P < 0.01.

Article Snippet: Three small interfering RNAs (siRNAs) targeting NRF2 (si-NRF2-1, si-NRF2-2, and si-NRF2-3) or HDAC2 (si-HDAC2-1, si-HDAC2-2, and si-HDAC2-3) were synthesized by GenePharma (Shanghai, China) for the silencing of each target, and their sequences are shown in .

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

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Effects of radiocontrast on nuclear factor, erythroid 2 like 2 (Nrf2) and cleaved caspase 3 expression. Representative western blot analyses of Nrf2 and cleaved caspase 3 expression over time after iohexol treatment and quantitative analyses are shown: in vitro ( A , B ) and in vivo ( C , D ) experiments. The expression of Nrf2 was significantly increased after iohexol treatment. Cropped gels are used in the figure, and the full-size gels are presented in Supplementary Figures – . * p < 0.05 vs . Cont and # p < 0.05 vs . CM 1 h. Abbreviations: Cont, saline control group; CM, iohexol treatment group.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: Expressing, Western Blot, In Vitro, In Vivo

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Changes in creatinine levels, histologic findings in the kidneys and renal injury markers after iohexol treatment. ( A ) Increased serum creatinine levels after iohexol treatment were aggravated by the loss of Nrf2 function. *p < 0.05 vs . WTcon and KOcon, # p < 0.05 vs . WT_CM. ( B ) Representative histology of the tubular changes after contrast injury. Iohexol treatment in mice induced tubular dilatation and vacuolization with tubular structure disruption. The severity and extent of tubular injury were aggravated upon the loss of Nrf2 (periodic acid-Schiff (PAS) staining; original magnification, ×400). ( C ) Quantitative measurement of tubular injury in histology by measuring the area of tubular dilatation and vacuolization. The area of tubular injury after iohexol treatment was significantly increased in the group with Nrf2 loss compared to that of the other groups. *p < 0.05 vs. WTcon and KOcon, # p < 0.05 vs . WT_CM. ( D ) Immunohistochemical staining of NGAL. Iohexol treatment in mice increased NGAL staining area and intensity in tubules, and these effects were further aggravated by the loss of Nrf2 (NGAL; Original magnification, ×400). ( E ) The area of NGAL positive staining was calculated and compared between groups. The histology of iohexol-treated Nrf2 KO mice showed significantly increased NGAL staining in renal tubules compared to that of the other groups. *p < 0.05 vs. WTcon and KOcon, # p < 0.05 vs . WT_CM. Abbreviations: WTcon, wild-type control group; KOcon, Nrf2 knockout control group; WT_CM, wild-type iohexol-treated group; KO_CM, Nrf2 knockout iohexol-treated group; Nrf2, nuclear factor, erythroid 2 like 2.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: Staining, Immunohistochemical staining, Knock-Out

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Cell cytotoxicity assays after iohexol treatment and Nrf2 inhibition. Nrf2 inhibition aggravated the decrease in cell viability measured by MTT assay after iohexol treatment. * p < 0.05 vs . Cont and siRNA, # p < 0 . 05 vs . CM. Abbreviations: Cont, control group; siRNA, siRNA transfection group; CM, iohexol-only treatment group, CM + siRNA, iohexol-treated group with Nrf2 inhibition by siRNA transfection; Nrf2, nuclear factor, erythroid 2 like 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: Inhibition, MTT Assay, Transfection

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Loss of Nrf2 in mice enhanced iohexol-induced apoptosis. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) staining of kidney tissue ( A ) and quantification of TUNEL-positive apoptotic nuclei (B) revealed an increase in apoptosis in renal tubules after iohexol treatment. In addition, this effect was aggravated by the loss of Nrf2. * p < 0.05 vs. WTcon and KOcon, # p < 0.05 vs. WT_CM. ( C ) Representative immunoblot analysis and quantitative analysis showed increased cleaved caspase 3 expression in tubular cells after iohexol treatment. Inhibition of Nrf2 enhanced cleaved caspase 3 expression. Cropped gels are used in the figure, and the full-size gels are presented in Supplementary Figure . * p < 0.05 vs . Cont and siRNA, # p < 0 . 05 vs . CM. Abbreviations: WTcon, wild-type control group; KOcon, Nrf2 knockout control group; WT_CM, wild-type iohexol-treated group; KO_CM, Nrf2 knockout iohexol-treated group; Cont, control group; siRNA, siRNA transfection group; CM, iohexol-only treatment group, CM + siRNA, iohexol treated group with Nrf2 inhibition by siRNA transfection; Nrf2, nuclear factor, erythroid 2 like 2.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: End Labeling, TUNEL Assay, Staining, Western Blot, Expressing, Inhibition, Knock-Out, Transfection

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Loss of Nrf2 enhanced the increase in iohexol-induced oxidative stress. ( A ) Oxidative stress in the kidneys determined by MDA concentration was increased after iohexol treatment, and this effect was augmented in mice with the loss of Nrf2 function. * p < 0.05 vs . WTcon and KOcon, # p < 0.05 vs . WT_CM. ( B ) ROS production according to DCF-DA staining was observed by confocal microscopy (2000× magnification). Green-colored immunofluorescence was increased after iohexol treatment, and this effect was enhanced by Nrf2 inhibition. ( C ) The percentage of fluorescence intensity of DCF-stained cells measured by a fluorescence spectrophotometer showed similar results. * p < 0.05 vs . Cont and siRNA, # p < 0 . 05 vs . CM 3 h and 6 h. Abbreviations: WTcon, wild-type control group; KOcon, Nrf2 knockout control group; WT_CM, wild-type iohexol-treated group; KO_CM, Nrf2 knockout iohexol-treated group; Cont, control group; siRNA, siRNA transfection group; CM, iohexol-only treatment group, CM + siRNA, iohexol-treated group with Nrf2 inhibition by siRNA transfection; Nrf2, nuclear factor, erythroid 2 like 2; DCF-DA, dichlorofluorescein diacetate.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: Concentration Assay, Staining, Confocal Microscopy, Immunofluorescence, Inhibition, Fluorescence, Spectrophotometry, Knock-Out, Transfection

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Immunoblot analyses of HO-1 and cytochrome c . An increase in HO-1 expression after iohexol treatment was not observed upon reduced Nrf2 function in both in vivo and in vitro experiments ( A ). Reduced Nrf2 function was also associated with enhanced cytochrome c expression. Cropped gels are used in the figure, and the full-size gels are presented in Supplementary Figure . ( B ). *p < 0.05 vs . Cont/siRNA or WTcon/KOcon. # p < 0.05 vs. CM or WT_CM. Cropped gels are used in the figure, and the full-size gels are presented in Supplementary Figure . Abbreviations: Cont, control group; siRNA, siRNA transfection group; CM, iohexol-only treated group, CM + siRNA, iohexol-treated group with Nrf2 inhibition by siRNA transfection; WTcon, wild-type control group; KOcon, Nrf2 knockout control group; WT_CM, wild-type iohexol-treated group; KO_CM, Nrf2 knockout iohexol-treated group; Nrf2, nuclear factor, erythroid 2 like 2.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: Western Blot, Expressing, In Vivo, In Vitro, Transfection, Inhibition, Knock-Out

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Loss of Nrf2 promotes macrophage infiltration in the renal tubules after iohexol treatment. Immunohistochemical staining of F4/80 ( A ) and quantitative analysis of F4/80 positive cells (red arrow, ( B ) showed increased macrophage infiltration after iohexol treatment, and these effects were augmented by the loss of Nrf2 function. * p < 0.05 vs . WTcon and KOcon, # p < 0.05 vs . WT_CM. Abbreviations: WTcon, wild-type control group; KOcon, Nrf2 knockout control group; WT_CM, wild-type iohexol-treated group; KO_CM, Nrf2 knockout iohexol-treated group; Nrf2, nuclear factor, erythroid 2 like 2.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: Immunohistochemical staining, Staining, Knock-Out

Journal: Scientific Reports

Article Title: The role of nuclear factor erythroid-2-related factor 2 expression in radiocontrast-induced nephropathy

doi: 10.1038/s41598-019-39534-2

Figure Lengend Snippet: Quantitative analysis of cell viability by 5-(3-carboxymethoxyphenyl)-2H-tetrazolium inner salt (MTS) assay after iohexol and Nrf2 activator (CDDO-Me) co-treatment with or without the inhibition of Nrf2 expression by siRNA transfection in NRK-52E cells. Cell viability was not changed upon pretreatment of CDDO itself. However, decreased cell viability after iohexol treatment was significantly attenuated by Nrf2 activation with 6-hour CDDO pretreatment. Nrf2 inhibition by siRNA pretreatment enhanced the decrease in cell viability after iohexol treatment, and CDDO pretreatment partially recovered the cell viability decrease induced by iohexol and Nrf2 inhibition * p < 0.05 vs . Con, CDDO, tcon, and tcon + CDDO, # p < 0.05 vs . CM, and † p < 0.05 vs . CM + CDDO, ‡ p < 0.05 vs . tCM.

Article Snippet: To assess the role of Nrf2 in CIN, Nrf2 expression in tubular cells was suppressed by transfection with a small interfering (si)RNA targeting Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). siRNA with complexed transfection agents (G-Fectin; Genolution, Seoul, Korea) was used as a negative control.

Techniques: MTS Assay, Inhibition, Expressing, Transfection, Activation Assay