Journal: PLoS ONE

Article Title: Disassembly of Shank and Homer Synaptic Clusters Is Driven by Soluble β-Amyloid 1-40 through Divergent NMDAR-Dependent Signalling Pathways

doi: 10.1371/journal.pone.0006011

Figure Lengend Snippet: Cultured cortical neurons were pre-treated for 45 min with the indicated pharmacological inhibitors before being treated for 1 h with Aβ (1 µM). Inhibitors were present in the medium during Aβ treatment. (A) PI-3K/mTOR pathway is required for Homer1b declustering. Pre-treatment with wortmannin (Wort, 2 µM) abolished the ability of Aβ to disperse Homer1b clusters (101.1±15.3%, cf. Aβ+Wort and Wort alone). Likewise, inhibition of the PI-3K downstream kinases PKB (with API-2, 30 µM) or mTor (with rapamycin, Rapa, 5 µM) prevented Aβ-induced declustering of Homer1b (90.6±8.4%, cf. Aβ+AP2 and API-2 alone; 95±8.2%, cf. Aβ+Rapa and rapamycin alone). Either the blockade of MEK/ERK pathway by pre-treatment with UO126 (10 µM; 70.9±4.8% UO126+Aβ vs UO126 alone) or cdk-5 inhibition by roscovitine (10 µM;63.7±5.8%, cf. Aβ+Rosco and Rosco alone) pre-treatment did affect Aβ-induced Homer declustering. (B) MEK/ERK pathway, but not PI-3K pathway, is required for Aβ-induced dispersal of Shank1. Pre-treatment with the PI-3K inhibitor wortmannin before Aβ treatment did not affect Aβ-induced Shank cluster dispersal (64.7±10.3%, cf. Wort+Aβ and Wort alone; p<0.05). Notably, wortmannin itself decreased Shank1 (but not Homer1b) cluster size (74.3±10.3%, cf. Wort and vehicle; p<0.05). Two structurally unrelated MEK inhibitors, UO126 (10 µM) and PD98059 (25 µM) prevented Aβ-induced declustering of Shank1 (96.8±12.1%, cf. PD98059+Aβ and PD98059 alone; 92.2±15.4%, cf. UO126+Aβ and UO126 alone). Likewise, the blockade of the Erk targeted kinase RSK by pre-treatment with SL0101-1 blocked Aβ effects (100.4±9.8%, p>0.05). Pre-treatment of neurons with the specific cdk-5 inhibitor roscovitine (Rosco, 10 µM) did not interfere with the ability of Aβ to reduce the size of Shank1 (72.6±10.8%, cf. Rosco+Aβ and Aβ alone) clusters. (C,D) PP2B is required for the dispersal of Homer1b, but not Shank1, clusters by Aβ. Pre-treatment with structurally unrelated PP2B inhibitors FK506 (10 µM) or cyclosporin (Cyclo, 100 µM) significantly blocked (p<0.05) Aβ-induced Homer1b declustering (98.8±10.4%, comparing FK506+Aβ vs. Aβ alone; 83.4±5.8%, comparing Aβ+Cyclo and Cyclo alone; and 61±7.9% in vehicle+Aβ-treated cells; see panel C ); however, all PP2B inhibitors were ineffective in preventing Shank1 declustering after Aβ exposure (61.3±4.5%, cf. FK506+Aβ and FK506 alone; 90.8±12.4%, cf. Cyclo+Aβ and Cyclo alone, see panel D ). For each condition, 8–12 neurons from 3–4 replicate experiments were considered (N = 8–12), and at least 50 synapses on each neuron were evaluated (on average, n = 500 puncta/condition).

Article Snippet: Nifedipine, (±)-verapamil, roscovitine, NiCl 2 , cycloheximide, cyclosporin and FK506 were purchased from Sigma Chemicals (Deisenhofen, Germany); NMDA, Bay-K4688, SL0101-1, MK801, UO126, PD98059, API-2, wortmannin, cantharidine were from Tocris (Bristol, UK); and sodium orthovanadate, rapamycin, TDZT, SU6656 and MG132 were from Calbiochem (La Jolla, CA).

Techniques: Cell Culture, Inhibition

Journal: Shock

Article Title: Inhibition of PAR-2 Attenuates Neuroinflammation and Improves Short-Term Neurocognitive Functions Via ERK1/2 Signaling Following Asphyxia-Induced Cardiac Arrest in Rats

doi: 10.1097/shk.0000000000001516

Figure Lengend Snippet: FIG. 5. Inhibition of ERK1/2 abolished the neuroinflammatory effect of PAR-2 activation at 24 h after ACA. Representative Western blot images (A) and quantitative analysis of PAR-2 (B), ERK1/2 (C), p-ERK1/2 (D), IL-6 (E), and TNF- a (F) in the brain revealed increased protein levels at 24 h following ACA except for ERK1/2 compared to the Sham group. Treatment with FSLLRY-NH2 significantly reduced p-ERK1/2 and proinflammatory cytokine levels compared to the ACA þ vehicle group. Further activation of PAR-2 with AC55541 only aggravated the neuroinflammatory response by increasing p-ERK1/2 expression. Potent ERK1/2 inhibitor PD98059 reversed the neuroinflammatory effect of AC55541. Neurologic outcome assessment with NDS at 24 h following ACA (G) revealed that the inhibition of PAR-2 significantly improved neurologic function while AC55541 alone significantly worsened performance compared to the ACA þ vehicle group. This detrimental effect of AC55541 on NDS was reversed by the ERK1/2 inhibitor, PD98059. Data are expressed as mean SD. n ¼ 6/group. ANOVA, Tukey. **P < 0.001 vs. Sham group, *P < 0.05 vs. Sham group, #P < 0.05 vs. ACA þvehicle group, &&P < 0.001 compared to ACA þ AC55541 group, &P < 0.05 compared to ACA þ AC55541 group. ACA indicates asphyxial cardiac arrest.

Article Snippet: Selective PAR-2 activator AC55541 (30 mg/rat) and ERK1/2 inhibitor PD98059 (Santa Cruz Biotechnology, Dallas, Tex; 2 mL of 2 mmol/L) were used for intervention (25).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

doi: 10.3389/fimmu.2018.01517

Figure Lengend Snippet: Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

Article Snippet: To assess PAR-induced cell signaling, starved cells were treated with 0–20 µM of either PAR-1, PAR-2, or PAR-4 antagonists (Peptides International, Louisville, KY, USA) for 30 min before stimulation with FVIIa + FX or FVIIa + FX + FII at indicated doses; or cells were stimulated with 0–100 µM of PAR-1, PAR-2, or PAR-4 agonist (Peptides International); or cells were treated first with or without 50 µM mitogen-activated protein kinase inhibitor PD98059, 10 µM p38-MAPK inhibitor SB203580, 20 µM NF-kB inhibitor SN50, and 1 µM of the S6K1 inhibitor (All from Merck Millipore, Hertfordshire, UK) for 30 min and then stimulated with 10 µM of PAR-1, PAR-2, or PAR-4 agonist.

Techniques: Coagulation, Isolation, Incubation, Functional Assay, Purification, Dissection, MANN-WHITNEY

Journal: International Journal of Biological Sciences

Article Title: Tail Fin Regeneration in Zebrafish: The Role of Non-canonical Crosstalk Between STAT3 and Vitamin D Pathway

doi: 10.7150/ijbs.96400

Figure Lengend Snippet: STAT3 phosphorylations regulate regeneration in vitro and in vivo . A Representative pictures, measurements of scratched area and levels of CXCL1 in supernatants of L929 cells after 6 hours of treatment with vehicle and 1 ng/ml IL-6. B Representative pictures, measurements of scratched area and levels of CXCL1 in supernatants of L929 cells after 24 hours of treatment with vehicle, 5 μM AZD1480 or 12.5 μM PD98059. C Representative pictures and quantification of regenerated area of wild type zebrafish tail fins cut at 3-dpf and treated for 2 days either with vehicle or 0.5 ng/ml IL-6. D Representative pictures and quantification of regenerated area of wild type zebrafish tail fins cut at 3-dpf and treated for 2 days either with vehicle or 200 nM LIF. E Representative pictures and quantification of regenerated area of stat3 +/+ , stat3 +/- and stat3 -/- tail fins cut at 3-dpf and analysed at 3 dpa. F Representative pictures and quantification of regenerated area of wild type zebrafish fins cut at 3-dpf and treated for 3 days either with DMSO, 50 μM AG490 or 12.5 μM PD98059. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Treatments of zebrafish larvae and cells were performed, at the concentration below reported, with the following compounds: LIF (H17002 Sigma); AG490 (T3434 Sigma); PD98059 (Axon 1386, Axon Medchem); vitamin D (D1530 Sigma); AZD1480 (Selleckchem, Houston, TX); IL-6 (R&D Systems).

Techniques: In Vitro, In Vivo