Cid755673

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.

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 NaHCO_3, 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).

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.