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9 protocols using cid755673

1

Evaluating Inhibitors of Signaling Pathways

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Rottlerin, an inhibitor of PKC (Calbiochem, La Jolla, CA), CID755673, an inhibitor of PKD (Tocris Biosciences, Ellisville, MO), Bay11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenenitrile], an inhibitor of IkB-α phosphorylation (Sigma, St. Louis, MO) and fludarabine, an inhibitor of STAT1 (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the medium at various concentrations between 0 and 100 µM in DMSO (Sigma, St. Louis, MO). DMSO was used as a vehicle control.
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2

Palmitate-induced β-cell dysfunction

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Human EndoC-βH1 cells were cultured as previously described (22 (link)). Mouse insulinoma (MIN6) cells were cultured in 25 mmol/L glucose DMEM supplemented with 15% FBS. Palmitate (sodium salt, Sigma-Aldrich) exposure media were supplemented with 2% fatty acid free BSA (Roche). During incubations with Palmitate serum-free medium was used for MIN6 cells. KRBH buffer contained 115 mmol/L NaCl, 24 mmol/L NaHCO3, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 0.2% BSA, and 10 mmol/L HEPES. Human pancreatic islets were kindly provided by Professor Olle Korsgren (Department of Radiology, Oncology and Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden), through the Uppsala facility for the isolation of human islets from Scandinavian brain-dead individuals. After isolation, the islets were cultured free-floating in Sterilin dishes in CMRL 1066 medium (ICN Biomedicals, Costa Mesa, CA, USA) containing 5.6 mmol/L glucose, 10% fetal calf serum, and 2 mmol/L L-glutamine for 1–5 days, and then subsequently transferred to the same culture conditions as those used for Palmitate exposure of EndoC-βH1 cells. All cells were kept at 37 °C in a humidified atmosphere with 5% CO2.
Etomoxir was from Sigma-Aldrich. GPR40 antagonist (GW1100) was from Calbiochem. The PKC inhibitor Bisindolylmaleimide (GF109203X) and the PKD inhibitor CID755673 were from Tocris Bioscience (Bristol, UK).
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3

CLL B Cell Purification and Stimulation

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CLL blood samples were obtained from untreated patients, after informed consent and validation by the local research ethics committee from the Avicenne Hospital (Bobigny, France), in accordance with the Declaration of Helsinki. CLL B cells were purified by negative selection using RosetteSep Human B Cell Enrichment Cocktail (StemCell Technologies, Grenoble, France). Purity (98,71% ± 1,41) was assessed as previously described [7 (link)]. CLL B cells were cultured fresh or after viable thawing in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (PAA, Les Mureaux, France). For BCR stimulation, CLL B cells (4×106 cells/well/12-well plate) were incubated with immobilized rabbit anti-IgM antibody (10 μg/mL; Jackson Immunoresearch, Montlucon, France) and incubated or not with Gö6976, Gö6983 (Calbiochem, Saint-Quentin-en-Yvellines, France), GF109203X, LY294002 (Sigma-Aldrich, Saint-Quentin-Fallavier, France), CAL101 (Selleckchem, Souffelweyersheim, France), CID755673 (Tocris Bioscience, Lille, France) or phorbol 12-myristate 13-acetate (PMA) (Cell Signaling Technology, Saint-Quentin-Fallavier, France). Human 293T and HepG2 cell lines were maintained in DMEM supplemented with 10% FBS.
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4

Signaling Pathway Reagents and Antibodies

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SDF-1α and CSF-1 were purchased from R&D Systems (Minneapolis, MN) and Pepro Tech (Rocky Hill, NJ), respectively. Gö 6976, Gö 6983, and U73122 were purchased from Calbiochem (Billerica, MA). CID755673 was purchased from Tocris (United Kingdom). fMLP and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies anti–phospho-PKD/PKCmu(S744/748), anti–phospho-PKD1(Ser-916), and anti-PKD/PKCμ were from Cell Signaling (Danvers, MA). Anti-SSH2 antibody was from Novus Biologicals. Anti–14‑3‑3 antibody was from Santa Cruz Biotechnology. Anti-GFP antibody was from BD Bioscience (San Jose, CA). Horseradish peroxidase (HRP)–conjugated anti-actin was from Santa Cruz Biotechnology (Dallas, TX). HRP-conjugated anti-mouse or anti-rabbit immunoglobulin G was obtained from Jackson Immuno­Research (United Kingdom). All tissue culture reagents were purchased from Invitrogen.
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5

Characterization of PKD Signaling Pathways

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CCK was from American Peptide (Sunnyvale, CA); Medium 199 was from GIBCO (Grand Island, NY). ATP and [γ-32P] ATP were from Perkin Elmer (Torrance, CA). CRT0066101 and CID755673 were obtained from TOCRIS (Mo, USA). Nitrocellulose membranes were from Schleicher and Schuell BioSience. Carbachol and GF1 (also known as GF109203X or bisindolylmaleimide I) were from Calbiochem (La Jolla, CA). Antibodies against PKD C-20, IκB-α, or LDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphoserine 744/748 PKD antibody that detects primarily the phosphorylated state of Ser 744 (Jacamo et al., 2008 (link)), phosphoserine 916 PKD antibody, antibodies for NF-κB P65, phosphoserine 32/36 IκB-α, GAPDH, ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). IL-6 antibody was from PeproTech (Rocky Hill, NJ) and MCP-1 antibody was from Antibodies-Online Inc. (Secaucus, NJ). Protein-A-agarose was from Roche Applied Science (Mannheim, Germany) and PKD substrate syntide-2, was from Bachem (Chicago, IL). Other items were from standard suppliers or as indicated in text.
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6

Molecular Mechanisms of Neuroprotection

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1-Methyl-4-phenyl pyridinium iodide (MPP+ iodide) and anti-β-actin antibody were purchased from Sigma-Aldrich (St. Lois, MO). The Bradford protein assay kit was purchased from Bio-Rad. Antibodies against PKD1, α-tubulin, and mtTFA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho PKD1 (p-S744/748 and p-S916), phospho-Akt (p-S473), total Akt, phospho-AMPKα (p-Thr172) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti-TH antibody was purchased from Millipore (Billerica, MA). The PKD1 inhibitor CID755673 was purchased from Tocris Bioscience (Ellisville, MO). Alexa 680-conjugated anti-mouse secondary antibody, MitoTracker Red FM, and all cell culture reagents were obtained from Invitrogen (Carlsbad, CA). IRDye 800-conjugated anti-rabbit secondary was purchased from Rockland Labs (Gilbertsville, PA).
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7

Pharmacological Modulation of GPCR Signaling

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The MOR selective agonist DAMGO (Tocris Bioscience, Ellisville, MO, USA), The MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) (Tocris Bioscience), and morphine (Sigma-Aldrich, St. Louis, MO, USA) were suspended in sterile serum-free RPMI-1640 medium, and stored at −80°C prior to use. H-89 (InvivoGen, San Diego, CA, USA), LY294002 (Sigma-Aldrich, St. Louis, MO, USA), Wortmannin (Sigma-Aldrich), U0126 (InvivoGen), CID755673 (Tocris Bioscience), U73122 (Sigma-Aldrich), Bay 43-9006 (Cayman, Ann Arbor, MI, USA), farnesylthiosalicytic acid (FTS) (Cayman), and Go 6983 (Sigma-Aldrich), were suspended in DMSO and stored at −20°C until use.
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8

Cardiomyocyte Contractility Assessment with ICL1-9

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% mean peak values of myocyte shortening were calculated from 6 consecutive peak shortening measurements at baseline and following addition of isoproterenol (ISO, 0.1 μM, Sigma-Aldrich) or ICL1-9 (10 μM, Palm-TAIAKFERLQTVTNYFIT-NH 2 , Peptide 2.0) (Fig. 1). For inhibitor assays, the myocytes were incubated with vehicle (0.1% DMSO) or PD184352 (10 μM, Sigma-Aldrich), Y-27632 (10 μM, Sigma-Aldrich), or ML7 (10 μM, Sigma-Aldrich) for 5 min or vehicle (0.1% DMSO), or CID755673 (10 or 25 μM, TOCRIS) for 45 min prior to baseline measurements and addition of either ISO or ICL1-9. Δ contractility (Table 1) was calculated as the difference between the % mean peak myocyte shortening values of ICL1-9-and Vehicle-treated myocytes.
Open in a separate window Figure 1: ICL1-9 induces cardiomyocyte contractility.
Representative cell length (μm) tracings of WT cardiomyocyte shortening at baseline or following addition of 0.1 μM ISO or 10 μM ICL1-9, each of which promoted significant contraction. The data are represented by the mean ± SEM from n = 4 (ISO) and 11 (ICL1-9) individual cardiomyocytes. ns = not significant, ***P<0.001, ****P<0.0001, one-way ANOVA with Tukey's multiple comparison test.
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9

Measuring Myocyte Contractility Modulation

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% mean peak values of myocyte shortening were calculated from 6 consecutive peak shortening measurements at baseline and following addition of isoproterenol (ISO, 0.1 μM, Sigma-Aldrich) or ICL1–9 (10 μM, Palm-TAIAKFERLQTVTNYFIT-NH2, Peptide 2.0) (Fig. 1). For inhibitor assays, the myocytes were incubated with vehicle (0.1% DMSO) or PD184352 (10 μM, Sigma-Aldrich), Y-27632 (10 μM, Sigma-Aldrich), or ML7 (10 μM, Sigma-Aldrich) for 5 min or vehicle (0.1% DMSO), or CID755673 (10 or 25 μM, TOCRIS) for 45 min prior to baseline measurements and addition of either ISO or ICL1-9. Δ contractility (Table 1) was calculated as the difference between the % mean peak myocyte shortening values of ICL1-9- and Vehicle-treated myocytes.
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