For differentiation, 2 day post confluent cells were incubated for 48 h in DMEM with 10% FBS, antibiotics, and a differentiation cocktail termed MDI, which contained 0. 5 mM isobutyl methylxanthine, 1 uM dexamethasone, and 1 ugml in sulin. click this After 48 h, the cells were maintained in DMEM with 10% FBS, antibiotics, and 5 ugml insulin. Cells were cultured for 12 days in DMEM with 10% FBS and antibiotics, and the media changed every 2 days until the cells were collected for analysis. Cytotoxicity assay Cytotoxicity was evaluated in vitro by determining cell viability using the CCK 8 kit. Cells were plated at a density of 1 103 cellsml in 96 well plates and allowed to attach overnight. The cells were then treated with POCU1b and incubated for 5 and 12 days.

After a 4 h incubation with the CCK 8 solution, absorbance was measured with a microtiter plate reader at 450 nm. We calculated the percent viability as optical density of treated sample optical density of untreated Inhibitors,Modulators,Libraries control Inhibitors,Modulators,Libraries 100. Oil Red O staining for intracellular triglycerides Cells were washed twice with PBS on day 12 and fixed on dishes with 3% formaldehyde in PBS for 20 min. After two rinses with PBS, cells were incubated with an Oil red O solution and filtered through a 0. 22 um filter for 30 min. The monolayer was extensively washed with water to remove unbound dye. Representative images of treated cells were obtained with an Olympus microscope, equipped with an Olympus DP 70 cam era. Stained cells were air dried overnight and then dis solved in isopropanol. Inhibitors,Modulators,Libraries Absorbance was measured at 520 nm.

Glycerol 3 phosphate dehydrogenase activity assay Treated Inhibitors,Modulators,Libraries cell lysates were extracted and used to deter mine GPDH activity as described. Briefly, protein lysate was measured according to the bicinchoninic acid assay method, and the GPDH assay was per formed to assess the disappearance of NADH during the GPDH catalyzed reduction of dihydroxyactone phos phate under zero order conditions as described. Immunoblotting Immunoblotting was performed using a previously de scribed method. Cells were homogenized in a solution containing 250 mM sucrose, 1 mM ethylenediaminetetra acetic acid, 0. 1 mM phenylmethylsulfonyl Inhibitors,Modulators,Libraries fluoride, and 20 mM potassium phosphate buffer. Equal amounts of protein were subjected to immunoblotting with the indicated antibodies. The antibodies used were the following.

PPAR, adipocyte differentiation necessary related protein, perilipin from Santa Cruz Biotechnology . pAMPK, and CEBP from Cell Signaling. The bound horseradish peroxidase conjugated secondary antibody was detected using an enhanced chemiluminescence detection system. Protein expression levels were determined by analyzing the signals captured on the nitrocellulose membranes using an image analyzer. RNA extraction and semi quantitative RT PCR Total RNA isolation and RT PCR were performed as previously described. For RT PCR, cDNA was syn thesized with 1 ug RNA using RT premix.