Journal: Frontiers in Pharmacology

Article Title: Andrographolide Protects against Aortic Banding-Induced Experimental Cardiac Hypertrophy by Inhibiting MAPKs Signaling

doi: 10.3389/fphar.2017.00808

Figure Lengend Snippet: Andr blocks MAPKs signaling in vivo and in vitro . (A) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, and P38 in the heart tissues of mice in the indicated groups ( n = 6). (B) Comparison of expression among the indicated groups. ∗ P < 0.05 compared with the corresponding sham group. # P < 0.05 vs. the veh-AB group. (C) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, and P38 in the cardiomyocytes in the indicated groups ( n = 6). (D) Comparison of expression among the indicated groups. ∗ P < 0.05 compared with the corresponding PBS group. # P < 0.05 vs. the vehicle-Ang II group.

Article Snippet: Cardiomyocytes were treated with ERK1/2 inhibitor (SCH772984, 5 μM, Selleck), JNK inhibitor (SP600125, 10 μM, Sigma), and/or P38 inhibitor (SB209063, 10 μM, Medchem Express) as well as stimulated with Ang II.

Techniques: In Vivo, In Vitro, Comparison, Expressing

Journal: Frontiers in Pharmacology

Article Title: Andrographolide Protects against Aortic Banding-Induced Experimental Cardiac Hypertrophy by Inhibiting MAPKs Signaling

doi: 10.3389/fphar.2017.00808

Figure Lengend Snippet: Andr-mediated cardioprotection depends on the inhibition of MAPKs in cardiomyocytes. Cardiomyocytes were treated with ERK1/2 inhibitor (SCH7729, 5 μM), JNK inhibitor (SP600125, 10 μM), and/or P38 inhibitor (SB209063, 10 μM) as well as stimulated with Ang II (1 μM) and treated with Andr (50 μM). (A) Cell viability was accessed by the cell counting kit-8 assay ( n = 5). (B–E) Immunofluorescence staining of α-actinin and the cell surface area of cardiomyocytes in the indicated groups ( n = 5 samples and n = 100+ cells per group). (F–J) The mRNA levels of ANP and β-MHC in cardiomyocytes in the indicated groups ( n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ∗ P < 0.05 compared with the control group. # P < 0.05 vs. the Ang II group.

Article Snippet: Cardiomyocytes were treated with ERK1/2 inhibitor (SCH772984, 5 μM, Selleck), JNK inhibitor (SP600125, 10 μM, Sigma), and/or P38 inhibitor (SB209063, 10 μM, Medchem Express) as well as stimulated with Ang II.

Techniques: Inhibition, Cell Counting, Immunofluorescence, Staining, Gene Expression, Control

Journal: Frontiers in Pharmacology

Article Title: Andrographolide Protects against Aortic Banding-Induced Experimental Cardiac Hypertrophy by Inhibiting MAPKs Signaling

doi: 10.3389/fphar.2017.00808

Figure Lengend Snippet: Andr reduces cardiac fibroblast activation via MAPKs. (A–F) Cardiac fibroblasts were treated with different concentrations of Andr (0, 12.5, 25, or 50 μM) and/or stimulated with Ang II (1 μM). (A) Cell viability was accessed by the cell counting kit-8 assay ( n = 5). (B) Cell proliferation was accessed by the cell counting kit-8 assay ( n = 5). (C) Immunofluorescence staining of α-SMA in the indicated groups. (D) The mRNA levels of collagen I, collagen III, and CTGF in cardiac fibroblasts in the indicated groups ( n = 6). (E) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, P38, and smad4 in the cardiac fibroblasts in the indicated groups ( n = 6). (F) Comparison of expression among the indicated groups. ∗ P < 0.05 compared with the corresponding PBS group. # P < 0.05 vs. the vehicle-Ang II group. (G–J) Cardiac fibroblasts were treated with ERK1/2 inhibitor (SCH7729, 5 μM), JNK inhibitor (SP600125, 10 μM), and/or P38 inhibitor (SB209063, 10 μM) as well as stimulated with Ang II (1 μM) and treated with Andr (50 μM). (G) Cell viability was accessed by the cell counting kit-8 assay ( n = 5). (H) Cell proliferation was accessed by the cell counting kit-8 assay ( n = 5). (I) Immunofluorescence staining of α-SMA in the indicated groups. (J,K) The mRNA levels of collagen I, collagen III, and CTGF in cardiac fibroblasts in the indicated groups ( n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ∗ P < 0.05 compared with the control group. # P < 0.05 vs. the Ang II group.

Article Snippet: Cardiomyocytes were treated with ERK1/2 inhibitor (SCH772984, 5 μM, Selleck), JNK inhibitor (SP600125, 10 μM, Sigma), and/or P38 inhibitor (SB209063, 10 μM, Medchem Express) as well as stimulated with Ang II.

Techniques: Activation Assay, Cell Counting, Immunofluorescence, Staining, Comparison, Expressing, Gene Expression, Control

Journal: PLoS ONE

Article Title: Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons

doi: 10.1371/journal.pone.0050138

Figure Lengend Snippet: Cells were pretreated with various of MAP kinase inhibitors SB203580 (10 µM), PD98059 (20 µM) or SP600125 (10 µM) for 30 min followed by stimulation with desipramine (20 µM; A) or fluoxetine (20 µM; D) for another 24 h. Whole cell lysates were subjected to Western blot for detection of HO-1 expression. Cells were incubated with desipramine or fluoxetine for the indicated time periods, and cell lysates were subjected to immunoblots with antibodies against phospho-JNK (B and C) and phospho-ERK (D and E). Results are the representatives of three or four independent experiments.

Article Snippet: SP600125 was obtained from Tocris Bioscience (Ellisville, MO).

Techniques: Western Blot, Expressing, Incubation

Journal: PLoS ONE

Article Title: Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons

doi: 10.1371/journal.pone.0050138

Figure Lengend Snippet: (A) Cells were treated with desipramine (20 µM) for indicated time periods (60 or 120 min) and nuclear extracts were collected, and the binding activity of Nrf2 to Nrf2-DNA binding element was examined by EMSA analysis. The DNA binding activity of Nrf2 is significantly different between desipramine treatment group and control group (one-way ANOVA followed by Bonferroni’s post hoc test). Cells were pretreated with PD98059 or SP600125 with desipramine (20 µM), and nuclear extracts were examined by EMSA analysis. Lane 1 was loaded without nuclear extracts (probe only). Results are expressed as the means ± S.E.M. from three independent experiments. *, p <0.05 as compared with the vehicle control group. #, p<0.05 as compared with the desipramine treatment group. (B) Cells were transfected with Control siRNA (100 nM) or Nrf2 siRNA (50 and 100 nM) for 24 h followed by stimulation with desipramine (20 µM) for another 24 h, and the protein levels of Nrf2 and HO-1 were determined by Western blot. The HO-1 expression is significantly different between Nrf2 siRNA group and control siRNA group (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, p <0.05 as compared with the control group. #, p<0.05 as compared with the desipramine treatment alone group.

Article Snippet: SP600125 was obtained from Tocris Bioscience (Ellisville, MO).

Techniques: Binding Assay, Activity Assay, Control, Transfection, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: HCV Core Protein Induces Chemokine CCL2 and CXCL10 Expression Through NF-κB Signaling Pathway in Macrophages

doi: 10.3389/fimmu.2021.654998

Figure Lengend Snippet: NF-κB signaling pathway plays pivotal role in CCL2 and CXCL10 expression in macrophages induced by HCV core protein. (A–E) MΦ-THP-1 were pretreated with specific inhibitor in different doses for 30 min and then treated with HCV core protein for 30 min. Cells were lysed and analyzed by immunoblotting with anti-p-NF-κB p65, anti-NF-κB p65, anti-p-AKT, anti-AKT, anti-p-JNK, anti-JNK, anti-p-p38, anti-p38, anti-p-ERK, anti-ERK and anti-GAPDH antibodies to determine the optimum concentration of inhibitors: IKKβ inhibitor IMD 0354 was 3 μM, Akt inhibitor MK 2206 2HCl was 5 μM, JNK inhibitor SP600125 was 20 μM, P38 inhibitor SB203580 was 30 μM, ERK inhibitor PD98059 was 50 μM. (F, G) MΦ-THP-1 were pretreated with optimal concentration of specific inhibitor and then treated with HCV core protein for 24 h. The supernatants were analyzed by the ELISA to detect CCL2 and CXCL10 production. (H, I) Luciferase activities were detected in HEK 293T cells co-transfected with NF-κB p65expressing plasmid, CCL2 or CXCL10 reporter plasmid and pRL-TK plasmid as described in Materials and Methods (co-transfected with promoter reporter plasmid and pRL-TK plasmid as control group). Data are presented as the relative ratio of Firefly luciferase activity to Renilla luciferase activity. Data are shown as mean ± SD for three independent experiments. ** p < 0.01.

Article Snippet: IKKβ inhibitor (IMD 0354), Akt inhibitor (MK 2206 2HCl), JNK inhibitor (SP600125), P38 inhibitor (SB203580) and ERK inhibitor (PD98059) were obtained from Selleckchem.

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Transfection, Plasmid Preparation, Control, Activity Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Roles of β-catenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells.

doi: 10.1038/labinvest.2011.144

Figure Lengend Snippet: Figure 4 Effects of Wnt3a on proliferation and gene expression in superficial zone (SFZ) cultures. SFZ cells were isolated from epiphyseal cartilage of wild-type mice and serially passaged at the density of 4200/cm2 up to six times. (a–f and i), The cultures were continuously treated with Wnt3a-containing conditioned medium (30%) or control conditioned medium after the first passage in the presence or absence of 100 ng/ml of DKK-1. Phase contrast pictures were taken on day 4 in the second passage cultures (a–d). Cell numbers were counted at indicated passages; values are average and s.d. from three independent samples (e). Total RNAs were prepared from Wnt3a-treated or control SFZ cell cultures at indicated passages to analyze expression levels of Proteoglycan 4 (Prg4), Ets-related gene (Erg), CD105 and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) by RT-PCR as described in Materials and Methods (f). Sixth passage cultures, treated with or without Wnt3a, were transferred into pellet cultures and cultured for 7 days. Total RNAs were then prepared to analyze expression level of aggrecan (Acan) or Prg4 (i). (g) The SFZ cells were transfected with Topflash Wnt reporter plasmid, treated with Wnt3a-containing conditioned medium (30%) or control conditioned medium in the presence or absence of 6BIO (BIO, 1 mg/ml), 100 ng/ml of DKK-1 or 10 mM IWR-1 for 24 h, and then the luciferase activity was measured. (h) The cultures were continuously treated with Wnt3a-containing conditioned medium (30%) or control conditioned medium after 1st passage in the presence or absence of 6BIO (BIO, 1 mg/ml), 100 ng/ml of DKK-1, 10 mM IWR-1, 10 mM SP600125 (JNK inhibitor) or 10 mM KN-93 (Ca2 þ /calmodulin-dependent protein kinase II inhibitor, CaMK inhibitor). Total RNAs were prepared from the SFZ cultures at passage 6 to analyze expression levels of Prg4 by real-time PCR. *Po0.05.

Article Snippet: Chondrocytes were isolated by additional overnight collagenase digestion of residual epiphyseal cartilage tissue as previously described.27 Cultures were treated with recombinant mouse Wnt3a (rWnt3a) (Chemicon, Temecula, CA, USA), conditioned medium containing Wnt3a,27 Wnt/b-catenin signaling inhibitors (recombinant mouse Dkk-1 (R&D systems), IWR-1-endo (Santa Cruz Biotechnology), KN-93 (an effective inhibitor of Ca2þ / calmodulin-dependent protein kinase II, Santa Cruz Biotechnology) or SP600125 (a selective inhibitor of c-Jun N-terminal kinases, Santa Cruz Biotechnology).

Techniques: Gene Expression, Isolation, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Immunology Author Choice

Article Title: Resolvin D1 Improves the Resolution of Inflammation via Activating NF-κB p50/p50–Mediated Cyclooxygenase-2 Expression in Acute Respiratory Distress Syndrome

doi: 10.4049/jimmunol.1700315

Figure Lengend Snippet: NF-κB is responsible for promoting the proresolving COX-2 protein expression in pulmonary fibroblasts. Primary pulmonary fibroblasts were incubated with 1 μg/ml LPS (L) for 24 h followed by administration of 100 nM RvD1 or vehicle (0.1% ethanol) for an additional 24 h, and 10 μM SP600125 (a JNK inhibitor), 10 μM SB203580 (a p38 MAPK inhibitor), and 10 μM MG-132 (an NF-κB inhibitor) were added 30 min prior to RvD1 administration. After incubation, the cells were harvested and sonicated. The expression of COX-2 was determined via Western blot. Cells were differentiated from lung tissues harvested from six rats per condition; n = 8 per treatment per group. Data are shown as mean ± SEM and are representative of at least four independent experiments. **p < 0.01, ***p < 0.001.

Article Snippet: After 24 h of culture with LPS (1 μg/ml) or a control medium, fibroblasts were treated with 100 nM RvD1 or a vehicle solution (0.1% ethanol, as the RvD1 was supplied in ethanol) for an additional 24 h. SP600125 (10 μM), SB603580 (10 μM), MG-132 (10 μM), and BOC-2 (10 μM) were added 30 min prior to RvD1 administration.

Techniques: Expressing, Incubation, Sonication, Western Blot

Journal: PLoS ONE

Article Title: Evaluation of the anti-inflammatory effects of synthesised tanshinone I and isotanshinone I analogues in zebrafish

doi: 10.1371/journal.pone.0240231

Figure Lengend Snippet: For a ), tailfin transection was performed on transgenic zebrafish larvae, TgBAC(mpx : GFP)i114 (3 days post-fertilisation, dpf), which were subjected to compound treatments immediately: DMSO (0.5%), SP600125 (30 μM) and TI 1 and TIIA 2 (10, 25 μM as indicated in brackets). Neutrophil number at the site of injury was assessed at 6 hpi, by counting GFP+ cells. For b ), tailfin transection was performed on zebrafish larvae (3 dpf), and at 4 hpi, good responders were subjected to compound treatments: DMSO (0.5%), TI 1 and TIIA 2 (10 μM, 25 μM as indicated in brackets). Neutrophil number at the site of injury was assessed at 8 hpi by counting GFP+ cells. For both graphs, data shown as mean ± SEM; n = 21–32 larvae per group from 3–4 independent experiments per graph. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to the DMSO vehicle control (one-way ANOVA with Dunnett’s multiple comparison post-test).

Article Snippet: The c-Jun N-terminal kinase inhibitor SP600125 was obtained from StressMarq Biosciences (Victoria, Canada), and tanshinone IIA (TIIA) 2 was obtained from Generon (Slough, UK).

Techniques: Transgenic Assay

Journal: PLoS ONE

Article Title: Evaluation of the anti-inflammatory effects of synthesised tanshinone I and isotanshinone I analogues in zebrafish

doi: 10.1371/journal.pone.0240231

Figure Lengend Snippet: a ) Structures of evaluated tanshinones (red), isotanshinones (blue) and the TI-acetal (green). b )- d ) Tailfin transection was performed on transgenic zebrafish larvae, TgBAC(mpx : GFP)i114 (3 dpf) which were subjected to compound treatments immediately: DMSO (0.5%), SP600125 (30 μM) and compounds 1 , 32–40 , 43 (25 μM). Neutrophil number at the site of injury was assessed at 6 hpi by counting GFP+ cells. Data shown as mean ± SEM; n = 30–39 larvae from 4 independent experiments per graph. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns not significant, in comparison to the DMSO vehicle control (one-way ANOVA with Dunnett’s multiple comparison post-test).

Article Snippet: The c-Jun N-terminal kinase inhibitor SP600125 was obtained from StressMarq Biosciences (Victoria, Canada), and tanshinone IIA (TIIA) 2 was obtained from Generon (Slough, UK).

Techniques: Transgenic Assay