repressive effects of palmitate are mediated by an increase in fatty acid-derived ceramide synthesis or fatty acid oxidation, the loss in stimulus-secretion coupling may involve a targeted disruption in calcium influx via voltage-dependent calcium channels in response to increased esterification/synthesis of fatty acid-derived metabo- lites such as DAG. Methods Reagents and antibodies Dulbeccos Modified Eagle Medium (DMEM), foetal bovine serum (FBS), Trypsin/EDTA solution and penicillin/streptomycin solution (containing penicillin G sodium (10,000 unit/ml), streptomycin sulphate (10,000 mg/ml) in 0.85% saline) were purchased from Gibco/Invitrogen Life Technologies (Paisley, UK). Other reagent grade chemicals were purchased from Sigma- Aldrich (Poole, UK). This included insulin, palmitate, C2- dihydroceramide, fenretinide, 2-bromopalmitate, dimethyl sulph- oxide (DMSO), 4,6-diamino-2-phenylindole diacetate (DAPI) and anti-actin. Bovine Serum Albumin (BSA) for conjugating palmitate and complete protein phosphatases inhibitor tablets were pur- chased from Boehringer-Roche Diagnostics (Basel, Switzerland). The Caspase-Glo 3/7 assay kit for apoptosis analysis was purchased from Promega (Southampton, UK). The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 b cells was from Mercodia (Uppsala, Sweden). C2- ceramide was purchased from Tocris (Bristol, UK). Antibodies to Protein Kinase B (PKB)/Akt, PKB/Akt phospho-Ser473 ERK phospho-Thr202/Tyr204, CaMKII phospho-Thr286 and CaMKII were purchased from New England Biolabs (Herts, UK) whilst that to atypical PKC l/f was from Santa Cruz (California, USA). The inhibitor AZ422 was synthesised and kindly supplied by Astra Zeneca (Cheshire, UK). Cell culture MIN6 b cells were grown to 80–90% confluence in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin. Cells were incubated for periods and with the appropriate effectors as indicated in the figure legends. Following appropriate incubation cells were washed twice with ice cold phosphate-buffered saline (PBS) prior to lysis in buffer containing 50 mM TRIS pH 7.4, 0.27 M sucrose, 1 mM Na- Orthovanodate pH 10, 1 mM EDTA 1 mM EGTA, 10 mM Na- b-glycerophosphate, 50 mM NaF, 5 mM Na-pyrophosphate, 1% (v/v) Triton X-100, 0.1% 2-mercaptoethanol and protease inhibitors. Whole cell lysates were centrifuged at 4000 g, 4uC for 10 min and stored at 220uC after being snap frozen in liquid nitrogen. Analysis of insulin release MIN6 b cells were serum starved for 1 h in Krebs Ringer Hepes (KRH) buffer (10 mM Hepes, pH 7.4, 129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2 and 0.1% (w/v) BSA) prior to the addition of KRH buffer+glucose (3 or 16 mM) or an amino acid mix containing a nominal amount of glucose (3 mM glucose, 10 mM leucine and glutamine) for a further 1 h before analysis. After appropriate cell incubations and stimulation, 100 ml of the supernatant was removed and transferred into two fresh 96 well plates. The media was then diluted 1:40 with KRH buffer (without BSA) and analysed for insulin content using an insulin ELISA kit. Unknown samples were calculated from a standard curve generated from known insulin standards supplied with the kit. Palmitate and Beta Cell Insulin Secretion Caspase-Glo 3/7 assay Apoptosis was assessed using a Caspase-Glo 3/7 assay kit from Promega. MIN6 b cells were seeded into a white-walled 96-well plate and incubated with the appropriate effectors. Cells were washed and 100 ml of serum free DMEM media added to each well. The Caspase-Glo substrate and buffer were mixed together thoroughly and 100 ml was then added to each well and left for up to 3 h. The luminescence generated by the assay is proportional to the amount of caspase activity present, allowing quantification of apoptotic activity. Analysis of cellular ceramide and DAG content After appropriate cell treatments, MIN6 b cells were extracted from plates using a rubber policeman in ice-cold PBS for analysis of ceramide and DAG using the DAG kinase method that relies on the kinase catalysing the formation of ceramide 1-phosphate and phosphatidic acid, respectively. Briefly, lipids were extracted from a cell aliquot containing 400 mg protein by the addition of chloroform/methanol (1:2, v/v) with the phases being broken by the addition of chloroform and 1 M NaCl. The organic phase was separated, dried and used as substrate for the DAG kinase assay for measurement of ceramide and DAG content as described previously [52]. SDS-PAGE and immunoblotting Cell lysates (30–50 mg protein) were reconstituted in Laemmli buffer and subjected to SDS-PAGE on 10% resolving gels and transferred onto immobilon-P membranes as described previously [53]. Membranes were probed with primary antibodies of interest and subsequently detected using either horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse-IgG (New England Biolabs). Membranes were visualized using enhanced chemiluminescence (Pierce, Northumberland, UK) on Konica Minolta x-ray medical film (Staffordshire, UK). 49-6-Diamidino-2-phenylindole (DAPI) staining of cell nuclei Cells were seeded onto 0.1 mm coverslips placed in a 6 well plate, and incubated with palmitate for times and at concentrations indicated in the figure legends. After the appropriate period of incubation with palmitate cells were washed three times with PBS. Cells were fixed by addition of 4% (w/v) paraformaldehyde (PFA) and left for 10 min. The PFA was removed and fixed cells washed three times with PBS before addition of 100 mM glycine for 15 min. The fixed cells were washed again with PBS three times and incubated with 50 ml of a diluted DAPI stock (10 mg/ml) for 15 min. The coverslips were then washed a further three times with PBS and mounted onto microscope slides. DAPI fluorescence was viewed and images captured using an inverted fluorescence microscope (Leica DM IRB, Leica, UK). Individual nuclei of adherent cells from randomly selected fields of view were counted from at least three separate experiments. Statistical analysis One way analysis of variance (ANOVA) followed by a Newman- Keuls post test, was used to analyse statistical significance. Data analysis was performed using GraphPad prism software and considered statistically significant at values of P,0.05. Supporting Information Figure S1 SPT is expressed in MIN6b cells and its activity is sensitive to AZ422. MIN6b cells were incubated in the absence and presence of 10 mM AZ422 for 48 h. Cells were PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e25975