Mouse preadipocyte (3T3-L1) and macrophage (RAW264.7) cells were purchased from the American Type Culture Collection (VA, USA) and then cultured as previously described12 (link),44 (link). Briefly, 3T3-L1 cells were normally differentiated into mature adipocytes until day 8, and in indirect co-culture, 3T3-L1 cell differentiation was induced in conditioned media from RAW264.7 cells. Oil red O staining of lipid droplets was used to monitor cell differentiation as previously described12 (link).
Human UCN3 gene cloned in pCMV6 vector tagged with Myc-DDK (OriGene, Rockville, MD, USA) was used for transfection. Myc-DDK tagged pCMV6 vector without UCN3 (OriGene, Rockville, MD, USA) was used as a control. 3T3-L1 adipocytes were transfected via electroporation using the Cell Line Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, 2 × 106 cells were electroporated with 2 μg of plasmid using Nucleofector program, A-033. About 24 h after transfection, the cells were treated with 400 µm of PA (Sigma-Aldrich, St. Louis, MA, USA) in low glucose (1 g/l) and 1% (w/v) serum medium for 24 h with and without insulin (20 nM for 10 min). PA was prepared in 10% (w/v) fatty acid free bovine serum albumin (BSA). The cells were then processed for RNA and protein analyses or plated for glucose uptake assay.
Human UCN3 gene cloned in pCMV6 vector tagged with Myc-DDK (OriGene, Rockville, MD, USA) was used for transfection. Myc-DDK tagged pCMV6 vector without UCN3 (OriGene, Rockville, MD, USA) was used as a control. 3T3-L1 adipocytes were transfected via electroporation using the Cell Line Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, 2 × 106 cells were electroporated with 2 μg of plasmid using Nucleofector program, A-033. About 24 h after transfection, the cells were treated with 400 µm of PA (Sigma-Aldrich, St. Louis, MA, USA) in low glucose (1 g/l) and 1% (w/v) serum medium for 24 h with and without insulin (20 nM for 10 min). PA was prepared in 10% (w/v) fatty acid free bovine serum albumin (BSA). The cells were then processed for RNA and protein analyses or plated for glucose uptake assay.
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