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: Oncotarget

Article Title: Laricitrin ameliorates lung cancer-mediated dendritic cell suppression by inhibiting signal transducer and activator of transcription 3

doi: 10.18632/oncotarget.13240

Figure Lengend Snippet: A. STAT3 inhibitor decreased lung cancer-mediated IL-10 expression. Laricitrin inhibited lung cancer-mediated upregulation on STAT3 activation B. and DNA binding activity C. in monocytes. Laricitrin inhibited LPS-mediated upregulation on STAT3 activation D. and DNA binding activity E. in monocytes. CD14 + monocytes were pretreated with laricitrin (2 μM) for 1 h, and then A549, CL1-5-CM or LPS (100 ng/ml) added for 30 min. The phosphorylation of STAT3 was assessed by immunoblot. The DNA binding activity of STAT3 was assessed by EMSA analysis. All results are representative of at least three independent experiments, and each value is the mean ± SD of three determinations. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The DNA binding activity of STAT3 transcription factor was examined using EMSA-based ELISA kits (Trans-AM STAT3 kit, Active Motif ® , Carlsbad, CA, USA).

Techniques: Expressing, Activation Assay, Binding Assay, Activity Assay, Western Blot

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition modulates the NF- κ B and CREB activation following CLP. Mice were subjected to CLP and treated with either SB216763 (25 mg/kg, i.p.) or vehicle (DMSO). Liver samples were harvested at 20 h after CLP. NF- κ B activity, as well as CREB activity, was assessed. Data are shown as mean ± SD. n = 6 per group. * P < 0.05, # P < 0.01 compared with vehicle-treated group.

Article Snippet: Levels of NF- κ B p65 and CREB activity in the nuclear extracts were quantified by TransAM NF- κ B p65 and TransAM CREB assay kits (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Inhibition, Activation Assay, Activity Assay

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition reduces the proinflammatory cytokine expression in cultured macrophages stimulated by LPS. (a) RAW264.7 cells were stimulated with LPS for 6 h in the absence or presence of SB216763 (10 μ M). TNF- α , IL-6, IL-1 β , and IL-10 mRNA expression levels were measured by quantitative PCR. (b) TNF- α and IL-6 concentration in the cultured medium was measured by ELISA. (c) RAW264.7 cells were stimulated with LPS for 1 h in the absence or presence of SB216763 (10 μ M). NF- κ B activity, as well as CREB activity, was assessed. Data are shown as mean ± SD. n = 5 per group. * P < 0.05, # P < 0.01 compared with vehicle-treated group.

Article Snippet: Levels of NF- κ B p65 and CREB activity in the nuclear extracts were quantified by TransAM NF- κ B p65 and TransAM CREB assay kits (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Inhibition, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay