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79952359734
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note
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2 environment. C3H/10T1/2 cells differentiated to mature adipocytes by the same medium containing 20 μg/ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) and 1 μM dexamethasone for four days, were replaced with medium containing 20 μg/ml insulin for three days, and then cultured for two days in culture medium.
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17
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79952362426
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note
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2 environment. Human subcutaneous preadipocytes differentiated to mature adipocytes by Adipocyte Differentiation Medium [Adipocyte medium, 0.25 mM IBMX, and a proprietary γ PPAR agonist (10 μM)] (Zen-Bio, Inc., #DM-2) for seven days, and then were replaced with fresh Adipocyte medium [DMEM/Ham′s F-12 (1:1, vol/vol), 3% fetal calf serum, 1 μM dexamethasone, 100 nM human insulin, 33 μM D-biotin, 17 μM Na-pantothenate, 15 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B] (Zen-Bio, Inc., #AM-1) for seven days.
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79952361307
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note
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4 cells/well in a 48-well plate) were incubated with compounds for 24 h at 37 °C and then glycerol assay was carried out. Cultured supernatants (25 μl) and 100 μl of free glycerol assay reagent (Cayman Chemical Company, MI) were mixed in a 96-well plate. After 15 min incubation at room temperature, the absorbance at 540 nm was measured with fluorescence plate reader (Bio-Tek Instruments, Inc., Winooski, VT). Absolute glycerol concentrations were calculated from a standard curve. Means and SEM are expressed as a percentage of the vehicle control.
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19
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79952363854
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note
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PDE 3B assays were performed in a reaction medium containing iFluor 488-AHC-cAMP, human recombinant PDE3B (BPS Bioscience Inc., San Diego, CA), 0.002% Brij, MgCl2, DTT and DMSO or compounds. This PDE3B activity assay was carried out in Caliper Life Sciences.
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