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: 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: International Journal of Molecular Sciences

Article Title: Identification of 7-Ketocholesterol-Modulated Pathways and Sterculic Acid Protective Effect in Retinal Pigmented Epithelium Cells by Using Genome-Wide Transcriptomic Analysis

doi: 10.3390/ijms24087459

Figure Lengend Snippet: Activation of JNK in response to 7KCh in mRPE cells. ( A ) Western blot estimation of phosphorylated JNK (p-JNK) levels in mRPE cells exposed to increasing concentrations of 7KCh for 24 h. ( B ) Cell viability determined by MTS assay in mRPE cells treated with 15 μM 7KCh and increasing concentrations of SP600125 (SP) for 24 h. ( C ) Secreted levels of IL-6, IL-8 and VEGF-A in mRPE cells exposed to 15 μM 7KCh and 40 μM SP600125 for 48 h and measured with ELISA. ( D ) Western blot estimation of p-JNK levels in mRPE cells exposed to 20 μM 7KCh and 10 μM SA for 6 h, 12 h and 24 h. In Western blot assays, JNK levels, previously normalized with respect to Actin, were used to normalize p-JNK quantification. In MTS assay and ELISA, the JNK inhibitor SP600125 was added with a pretreatment of 2 h with respect to 7KCh. The vehicle group in the graphs represents the control (control-vehicle) and the 7KCh (7KCh-vehicle) treatment. Data are represented as mean ± SEM of at least three different experiments. The dashed and dotted lines are a guidance mark of control and 7KCh value, respectively. ANOVA test was used for statistical analysis followed by Tukey (in A , B , D ) or Sidak (in C ) post hoc test.* p < 0.05; ** p < 0.01.

Article Snippet: Cells were treated with 8–20 μM 7KCh (Sigma-Aldrich, Madrid, Spain) alone or with 10 μM SA (PPQF, University of Alcalá, Madrid, Spain), 10 μM of the TLR4 inhibitor CLI-095 (Invivogen Inc., San Diego, CA, USA), 10–50 μM of the JNK inhibitor SP600125 (StressMarq Biosciences Inc., Victoria, BC, Canada) or 5–40 μM of the p38 inhibitor SB203580 (Sigma-Aldrich, Madrid, Spain).

Techniques: Activation Assay, Western Blot, MTS Assay, Enzyme-linked Immunosorbent Assay, Control

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