Mithras lb 940 apparatus

A reporter gene containing an upstream fragment of 2 kilobases of the 5′ promoter region in the human PRL gene linked to pGL3-basic reporter vector. ESRRG-cryptic (NM_001243518.1), ESRRG-canonical (NM_001243519.1) cDNA were, respectively, cloned into pCDH vector. Human Pit-1 cDNA (NM_000306.1) was respectively cloned into pCDNA3.1 vector. All constructs were verified by Sanger sequencing (TsingKe Biological Technology). HEK293 cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO_2. The culture medium was DMEM with 10% FBS. Cultures were fed every other day. The cell lines were also genotyped to rule out cross-contamination and their morphology was regularly examined. HEK293 cells were transfected in 24-well plates containing 2 μL of Lipofectamine 2000, 1 μg of pCDH-ESRRG-cryptic/pCDH-ESRRG-canonical and/or pCDNA3.1/ pCDNA3.1-Pit-1, 1 μg of pGL3-basic or pGL3-basic-pRL-p, and 100 ng of pRL-TK-Renilla (as transfection control) for 48 h. The cells were lysed in buffer (100 μL lysis buffer, Promega Corporation) and luciferase activity was then measured in a Mithras LB 940 apparatus (Berthold Technologies). The different vectors transfection groups are shown in Supplementary Table 12.

NF-κB luciferase reporter gene cells were generated by stably transfecting SH-SY5Y cells with the pGL4.32[luc2P/NF-κB-RE/Hygro] vector (Promega, Charbonnières les bains, France) containing five copies of an NF-κB response element that drives transcription of the luciferase reporter gene luc2P and a mammalian selectable marker for hygromycin resistance. After hygromycin selection (200 µg/mL), surviving cells were transfected with a control vector containing β-galactosidase with a cytomegalovirus promoter. 48 h after transfection, serum starved cells were treated 4 h with resistin (200 ng/ml), palmitic acid (200 µM) or DHA (20 µM) then luciferase and β-galactosidase activity were measured using the Mithras LB 940 apparatus (Berthold technologies, Bad Wildbad, Germany) and the Dual-Light Chemiluminescent Reporter Gene Assay System (Applied Biosystems, llkirch, France) according to the manufacturer’s instructions. Luciferase activity was normalized by β-galactosidase activity. All transfections were performed using Lipofectamine 2000 (Invitrogen, llkirch, France) according to the manufacturer’s instructions.