Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: (A and B) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours; pretreated with vehicle or sulforaphane (SFN) (2–4 μM) for 1 hour; and then incubated with serum-free medium or medium containing 2.5% fetal bovine serum (FBS). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation 48 hours after treatment (A). p21Waf1and p27Kip1 expression was determined 24 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (B). (C and D) ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with serum-free medium or medium containing 2.5% FBS. DNA synthesis was determined by measuring BrdU incorporation 72 hours after treatment (C). p21Waf1and p27Kip1 expression was determined 72 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (D). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle control or Ad-GFP. Bars represent mean ± SEM of six ASMC (A and C) and four ASMC donors (B and D).

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Incubation, DNA Synthesis, BrdU Incorporation Assay, Expressing, Western Blot, Infection

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: (A and B) Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours and then treated with transforming growth factor (TGF)-β (1 ng/ml) for 0.5–24 hours (A), or TGF-β (0.25–1 ng/ml) for 24 hours (B). Heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO1) mRNA was determined by real-time polymerase chain reaction (PCR) and normalized to 18S rRNA expression. (C and D) Confluent ASMCs were transfected with antioxidant response elements (ARE)–driven luciferase reporter vector for 18 hours, serum-deprived for 6 hours, and finally treated with TGF-β (0.25–1 ng/ml) for 20 hours (C) or pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour and then treated with TGF-β (0.25 ng/ml) for 20 hours (D). ARE-driven transcriptional activity was determined by measuring firefly luciferase activity and normalizing to Renilla luciferase activity. (E and F) Confluent ASMCs were serum-deprived for 24 hours and then treated with TGF-β (0.25–1 ng/ml) for 20 hours. Nuclear factor E2-related factor 2 (Nrf2) expression was determined in whole-cell extracts by Western blotting and normalized to β-actin expression (E). Nrf2-ARE binding was determined in nuclear extracts by an ELISA-based TransAM assay (F). Bars represent mean ± SEM of three ASMC (A and F), five ASMC (B), three to six ASMC (C), four ASMC (D), and four ASMC donors (E). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with unstimulated control. ns = nonsignificant.

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then stimulated with transforming growth factor (TGF)-β (0.25 ng/ml) for 24 hours (A–C). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 24 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (C–E). Heme oxygenase (HO)-1, catalase, and manganese superoxide dismutase (MnSOD) mRNA expression was determined by real-time polymerase chain reaction and normalized to 18S rRNA expression. Bars represent mean ± SEM of five ASMC (A–C) and four ASMC donors (C–E). *P < 0.05, **P < 0.01. ns = non-significant.

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Incubation, Expressing, Infection, Real-time Polymerase Chain Reaction

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: (A and B) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then incubated with medium containing 2.5% fetal bovine serum (FBS) or 2.5% FBS and transforming growth factor (TGF)-β (0.25 ng/ml) for 72 hours (A). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with medium containing 2.5% FBS or 2.5% FBS and TGF-β (0.25 ng/ml) for 72 hours (B). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation. (C–F) Confluent ASMCs were serum-deprived for 24 hours, pretreated with vehicle control or sulforaphane (2–4 μM) for 1 hour, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (C and D). Alternatively, ASMCs were incubated with Ad-GFP or Ad-Nrf2 (MOI 250) for 18 hours, serum-deprived for 6 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (E and F). IL-6 mRNA expression was determined by real-time polymerase chain reaction, normalized to 18S rRNA expression, and expressed as fold change with respect to unstimulated control. IL-6 release was determined by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle or Ad-GFP control. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with TGF-β and vehicle or Ad-GFP–treated cells. Bars represent mean ± SEM of five ASMC donors (B and C), four ASMC donors (A, D, and F), and three ASMC donors (E).

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Incubation, Expressing, Infection, DNA Synthesis, BrdU Incorporation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: Airway smooth muscle cells (ASMCs) cultured from bronchoscopic biopsies and transplant airways taken from healthy subjects (n = 5–6) or bronchoscopic biopsies from patients with nonsevere (n = 6) and severe asthma (n = 6–7) were grown to confluence in medium containing 10% fetal bovine serum, and whole-cell protein was extracted. (A and B) Nuclear factor E2-related factor 2 (Nrf2) protein expression was determined in whole-cell protein extracts by Western blotting and normalized to β-actin expression. To ensure that all membranes were equally exposed to antibodies, substrate, and X-ray film a control sample (c) was run in each of the gels. (C) Nrf2–antioxidant response elements (ARE) binding was determined in whole-cell protein extracts by an ELISA-based TransAM assay. Data were analyzed using Mann-Whitney test.

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Cell Culture, Expressing, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Antioxidants (Basel, Switzerland)

Article Title: Enhancing Bioactive Components of Euryale ferox with Lactobacillus curvatus to Reduce H 2 O 2 -Induced Oxidative Stress in Human Skin Fibroblasts.

doi: 10.3390/antiox11101881

Figure Lengend Snippet: Figure 9. Effects of UF and LCF on Nrf2/Keap1 signaling pathway. Effects of UF and LCF on Nrf2 content (a) and mRNA expression (b), and Keap1 content (c) and mRNA expression (d). Effects of UF and LCF on activity of NQO-1 (e) and mRNA expression (f), HO-1 content (g) and mRNA expression (h), and GCLC content (i) and mRNA expression (j). UF: unfermentation; LCF: single microbial fermentation with L. curvatus. Analysis of significant differences between each group and H2O2 model group, #: p < 0.05; ##: p < 0.01; ###: p < 0.001.; comparison of each group between UF and LCF, *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: p > 0.05.

Article Snippet: PBS, DMEM medium, newborn calf serum, streptomycin (100 mg/L), penicillin (1 × 105 U/L), and 0.25% trypsin (including EDTA) were purchased from GIBCO Life Technologies, Shanghai, China; ascorbic acid and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) were purchased from Alfa Aesar, China; Chemical Co., Ltd., Shanghai, China; Western and IP cell lysate, PMSF and ROS Detection Kits were purchased from Biyuntian Biotechnology Co., Ltd., Shanghai, China; CCK-8 Cell Proliferation and Toxicity Detection Kit (Biorigin (Beijing) Inc., Beijing, China; Total Glutathione Peroxidase (GSH-Px) Assay Kit with Nicotinamide Adenine Dinucleotide Phosphate (NADPH), Catalase Assay Kit, ROS Detection Kit and Cell Counting Kit, Total Antioxidant Capacity Test Kit (ABTS method) were purchased from Beyotime Biotechnology Co., Ltd., Shanghai, China; Human Elastase ELISA Kit, Human Collagen Type I ELISA Kit, Human Total Matrix Metalloproteinase 1 (MMP-1) ELISA Kit and Human NRF2 ELISA Kit were purchased from Cusabio (https://www.cusabio.cn/, accessed on 23 March 2022), Wuhan, China.

Techniques: Expressing, Activity Assay, Comparison

Journal: Antioxidants (Basel, Switzerland)

Article Title: Enhancing Bioactive Components of Euryale ferox with Lactobacillus curvatus to Reduce H 2 O 2 -Induced Oxidative Stress in Human Skin Fibroblasts.

doi: 10.3390/antiox11101881

Figure Lengend Snippet: Figure 13. The oxidative stress pathway of Nrf2/Keap1/Mafk.

Article Snippet: PBS, DMEM medium, newborn calf serum, streptomycin (100 mg/L), penicillin (1 × 105 U/L), and 0.25% trypsin (including EDTA) were purchased from GIBCO Life Technologies, Shanghai, China; ascorbic acid and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) were purchased from Alfa Aesar, China; Chemical Co., Ltd., Shanghai, China; Western and IP cell lysate, PMSF and ROS Detection Kits were purchased from Biyuntian Biotechnology Co., Ltd., Shanghai, China; CCK-8 Cell Proliferation and Toxicity Detection Kit (Biorigin (Beijing) Inc., Beijing, China; Total Glutathione Peroxidase (GSH-Px) Assay Kit with Nicotinamide Adenine Dinucleotide Phosphate (NADPH), Catalase Assay Kit, ROS Detection Kit and Cell Counting Kit, Total Antioxidant Capacity Test Kit (ABTS method) were purchased from Beyotime Biotechnology Co., Ltd., Shanghai, China; Human Elastase ELISA Kit, Human Collagen Type I ELISA Kit, Human Total Matrix Metalloproteinase 1 (MMP-1) ELISA Kit and Human NRF2 ELISA Kit were purchased from Cusabio (https://www.cusabio.cn/, accessed on 23 March 2022), Wuhan, China.

Techniques:

Journal: Journal of Nanobiotechnology

Article Title: Self-immolative nanocapsules precisely regulate depressive neuronal microenvironment for synergistic antidepression therapy

doi: 10.1186/s12951-023-02008-9

Figure Lengend Snippet: In vivo antidepressive effects of nanocapsules. A In vivo Cy7.5 fluorescence of C57BL/6J mice and CUMS mice after intravenous injection with VCNCs-Cy7.5, CNCs-Cy7.5, and VNCs-Cy7.5 at 400 μg 5-HT/kg for 3 h and 7 h. Living Cy 5.5 fluorescence images of mice after treatment with VCNCs-Cy5.5, CNCs-Cy5.5, and VNCs -Cy5.5 (C 5-HT : 400 μg /kg) for 3 h. The representative western blot analysis of B Nrf 2 in the hippocampus of brain across all groups. The other two replicates were presented in Additional file : Fig. S38. Reverse transcription quantitative polymerase chain reaction analysis (RT-qPCR) of relative mRNA levels of C NLRP3 in the hippocampi of mice in all groups (n = 3). The levels of hippocampal D IL-6 in the mice hippocampi after treatment were detected using ELISA kits. The results were normalized by the protein concentration of each sample (n = 3). E ROS/DAPI staining and F GFAP / DAPI staining brains in groups subjected to different treatments. Analysis of hippocampal BDNF expression using G western blot, H RT-qPCR, I ELISA kits and J immunohistochemistry slides. The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: The sample solutions were then measured using IL-6 (Elabscience, #E-MSEL-M0001, China), IL-1β (Elabscience, #E-EL-M0037c, China), TNF-α (Elabscience, #E-EL-M3063, China), BDNF (Elabscience, #E-EL-M0203c, China), Nrf2 (Cusabio, #CSB-E16188m, China), and 5-HT ELISA kits (Elabscience, #E-EL-0033c China) according to the manufacturer’s instructions.

Techniques: In Vivo, Fluorescence, Injection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Protein Concentration, Staining, Expressing, Immunohistochemistry

Journal: Journal of Nanobiotechnology

Article Title: Self-immolative nanocapsules precisely regulate depressive neuronal microenvironment for synergistic antidepression therapy

doi: 10.1186/s12951-023-02008-9

Figure Lengend Snippet: Effects of nanocapsules on in vivo 5-HT level and neuroprotection. A 5-HT IHC and B nissil and HE staining of brains after administering with fluoxetine, VCNCs, CNCs, and VNCs. C The levels of hippocampal 5-HT in the mice hippocampi after treatment were detected using ELISA kits. The results were normalized by the protein concentration of each sample (n = 3). The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05. D The in vivo therapeutic outcomes of nanocapsules

Article Snippet: The sample solutions were then measured using IL-6 (Elabscience, #E-MSEL-M0001, China), IL-1β (Elabscience, #E-EL-M0037c, China), TNF-α (Elabscience, #E-EL-M3063, China), BDNF (Elabscience, #E-EL-M0203c, China), Nrf2 (Cusabio, #CSB-E16188m, China), and 5-HT ELISA kits (Elabscience, #E-EL-0033c China) according to the manufacturer’s instructions.

Techniques: In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Iranian Journal of Basic Medical Sciences

Article Title: p-Coumaric acid protects cardiac function against lipopolysaccharide-induced acute lung injury by attenuation of oxidative stress

doi: 10.22038/ijbms.2019.36316.8650

Figure Lengend Snippet: Effects of p-CA on LPS-induced systemic inflammation. Nrf2 mRNA expression in heart tissue analyzed. Data are expressed as the mean±SEM (n=8). * P <0.05, versus the Control rats. # P <0.05 versus the LPS group. Nrf2: Nuclear factor-erythroid 2 -related factor 2; LPS: Lipopolysaccharide; p-CA: p-Coumaric acid

Article Snippet: Expression of Nrf2 gene RNeasy plus mini kit (Qiagen Co, Netherlands) was used for RNA extraction.

Techniques: Expressing, Control

Journal: Drug Design, Development and Therapy

Article Title: Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells

doi: 10.2147/dddt.s75221

Figure Lengend Snippet: Figure 13 ALS downregulates the expression level of Nrf2 in PANC-1 and BxPC-3 cells. Notes: Cells were treated with ALS at concentrations of 0.1 μM, 1 μM, and 5 μM for 24 hours and then protein samples were subject to Western blotting assay. (A) Representative blots of Nrf2 in PANC-1 and BxPC-3 cells. (B) Bar graphs showing the relative expression level of Nrf2 in PANC-1 and BxPC-3 cells. Data represent the mean ± SD of three independent experiments. *P0.05, **P0.01, and ***P0.001 by one-way ANOVA. Abbreviations: ALS, alisertib; Nrf2, nuclear factor (erythroid-derived 2)-like 2; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: Primary antibodies against human cyclin-dependent kinase (CDK) 1/CDC2, CDK2, cyclin B1, cyclin D1, p21 Waf1/ Cip1, p27 Kip1, p53, Sirt1, p38 MAPK, phosphorylated (p-) p38 MAPK at Thr180/Tyr182, AMPK, p-AMPK at Thr172, PI3K, p-PI3K/p85 at Tyr458, Akt, p-Akt at Ser473, mTOR, p-mTOR at Ser2448, p-p44/42 MAPK (Erk1/2) at Thr202/204, p-53 at Ser392, Ac-p53 at Thr288, phosphatase and tensin homolog (PTEN), beclin 1, microtubule-associated protein 1A/1B-light chain 3 (LC3)-I, LC3-II, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and Aurora (#3875) and EMT (#9782) antibody sampler kits were all purchased from Cell Signaling Technology Inc (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Derivative Assay, Standard Deviation