Ained from the Ambroise Pare ?Hospital (Paris, France).Cloning of the

Ained from the Ambroise Pare ?Ergocalciferol Hospital (Paris, France).Cloning of the Human LDHB Promoter and Construction of the Reporter PlasmidsLDHB promoter reporter plasmids were constructed using a human genomic DNA fragment from the 59-flanking region of the human LDHB promoter to the TSS as matrix. To generate the different constructions of the reporter plasmids 25033180 p.LDHB-Luc 1188, p.LDHB-Luc 611, p.LDHB-Luc 515 and p.LDHB-Luc 105, we amplified the LDHB promoter using the same reverse primer (59-AAGCTTCTACCAGGAGAGAGAAGGCT-39) and forward primers as follows: p.LDHB-Luc 1188: 59-AGATCTGGCACTGAGAATAAACTGAA-39, p.LDHB-Luc 611: 59-AGATCTCTGTAATCCCAGCACTTTGG-39, p.LDHB-Luc 515: 59-AGATCTCCCCTCTCTACTAAAAATAC-39, p.LDHB-Luc 105: 59-AGATCTTGAAGGGGATTGAGCGAG-39. PCR products were doubly digested with Bgl2 and HindIII and inserted into the pGL3-basic vector. The identity of the constructions was confirmed by sequencing.Cell CulturesThree human follicular thyroid carcinoma cell lines were used: the XTC.UC1 cells were oncocytic variants kindly provided by O. Clark [16], and the other cell lines, FTC-133 and RO82 W-1, were obtained from the Interlab Cell Line Collection (National Institute for Cancer Research, Genoa, Italy) and originated from classical follicular carcinomas. FTC-133 and XTC.UC1 cells were grown in Dulbecco’s modified medium (Invitrogen Corp., (-)-Calyculin A site Carlsbad, CA, USA), supplemented with 10 fetal bovine serum (Seromed, Biochrom AG, Berlin, Germany), 1 L-glutamine (Invitrogen, Carlsbad, CA, USA) and 1 penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). We added 10 mU/ml TSH (Sigma-Aldrich, Saint Louis, MO, USA) for XTC.UC1. RO82 W-1 cells were grown in 60 Dulbecco’s modified medium, and 30 endothelial basal medium (both from PAA, Pasching, Austria) supplemented with 10 fetal bovine, 1 Lglutamine, and 1 penicillin/streptomycin. For treatment with the inverse agonist XCT790 (Sigma-Aldrich, Saint Louis, MO, USA) was used a concentration validated for its specific ERRa inhibition in our cellular models [6]. FTC-133 and RO82W-1 cells were treated for 10 days with a final concentration of 5 mM XCT790, replaced with fresh media every three days.Transient Transfections and Luciferase AssayRO82W-1 cells were plated two days before transfection. Transient transfection was performed with lipofectamine (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer. Cells were collected 48 h later for functional and quantitative PCR analyses. According to the experiments, RO82W-1 cells were transfected with 1 mg LDHB promoter reporter plasmid (p.LDHB Luc), 0.05 mg of plasmid PRC (Origene Technologies, Rockville, MD, USA), 0.05 mg of plasmid ERRa (Addgene, Cambridge, MA, USA) and 0.5 mg of pRL-CMV (Promega, Madison, WI, USA) used as an internal control of transfection efficiency. For experimentation with luciferase activity, cells were harvested after 48 h of treatment for the luciferase reporter assay using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to that of the internal control, Renilla luciferase, used as the relative luciferase unit. All assays were done in duplicate in three separate experiments.Bioinformatics Analysis of LDH PromotersWe extracted LDHA and LDHB promoter sequences from nucleotides 22000 to 21 starting from the transcription starting site (TSS) according to the NCBI accession NM_00566 and NM_002300. We scanned the promoters with the Matrix-Scan software (http://rsat.ulb.ac.be/rsat/).Ained from the Ambroise Pare ?Hospital (Paris, France).Cloning of the Human LDHB Promoter and Construction of the Reporter PlasmidsLDHB promoter reporter plasmids were constructed using a human genomic DNA fragment from the 59-flanking region of the human LDHB promoter to the TSS as matrix. To generate the different constructions of the reporter plasmids 25033180 p.LDHB-Luc 1188, p.LDHB-Luc 611, p.LDHB-Luc 515 and p.LDHB-Luc 105, we amplified the LDHB promoter using the same reverse primer (59-AAGCTTCTACCAGGAGAGAGAAGGCT-39) and forward primers as follows: p.LDHB-Luc 1188: 59-AGATCTGGCACTGAGAATAAACTGAA-39, p.LDHB-Luc 611: 59-AGATCTCTGTAATCCCAGCACTTTGG-39, p.LDHB-Luc 515: 59-AGATCTCCCCTCTCTACTAAAAATAC-39, p.LDHB-Luc 105: 59-AGATCTTGAAGGGGATTGAGCGAG-39. PCR products were doubly digested with Bgl2 and HindIII and inserted into the pGL3-basic vector. The identity of the constructions was confirmed by sequencing.Cell CulturesThree human follicular thyroid carcinoma cell lines were used: the XTC.UC1 cells were oncocytic variants kindly provided by O. Clark [16], and the other cell lines, FTC-133 and RO82 W-1, were obtained from the Interlab Cell Line Collection (National Institute for Cancer Research, Genoa, Italy) and originated from classical follicular carcinomas. FTC-133 and XTC.UC1 cells were grown in Dulbecco’s modified medium (Invitrogen Corp., Carlsbad, CA, USA), supplemented with 10 fetal bovine serum (Seromed, Biochrom AG, Berlin, Germany), 1 L-glutamine (Invitrogen, Carlsbad, CA, USA) and 1 penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). We added 10 mU/ml TSH (Sigma-Aldrich, Saint Louis, MO, USA) for XTC.UC1. RO82 W-1 cells were grown in 60 Dulbecco’s modified medium, and 30 endothelial basal medium (both from PAA, Pasching, Austria) supplemented with 10 fetal bovine, 1 Lglutamine, and 1 penicillin/streptomycin. For treatment with the inverse agonist XCT790 (Sigma-Aldrich, Saint Louis, MO, USA) was used a concentration validated for its specific ERRa inhibition in our cellular models [6]. FTC-133 and RO82W-1 cells were treated for 10 days with a final concentration of 5 mM XCT790, replaced with fresh media every three days.Transient Transfections and Luciferase AssayRO82W-1 cells were plated two days before transfection. Transient transfection was performed with lipofectamine (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer. Cells were collected 48 h later for functional and quantitative PCR analyses. According to the experiments, RO82W-1 cells were transfected with 1 mg LDHB promoter reporter plasmid (p.LDHB Luc), 0.05 mg of plasmid PRC (Origene Technologies, Rockville, MD, USA), 0.05 mg of plasmid ERRa (Addgene, Cambridge, MA, USA) and 0.5 mg of pRL-CMV (Promega, Madison, WI, USA) used as an internal control of transfection efficiency. For experimentation with luciferase activity, cells were harvested after 48 h of treatment for the luciferase reporter assay using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to that of the internal control, Renilla luciferase, used as the relative luciferase unit. All assays were done in duplicate in three separate experiments.Bioinformatics Analysis of LDH PromotersWe extracted LDHA and LDHB promoter sequences from nucleotides 22000 to 21 starting from the transcription starting site (TSS) according to the NCBI accession NM_00566 and NM_002300. We scanned the promoters with the Matrix-Scan software (http://rsat.ulb.ac.be/rsat/).