Cancer du sein et micro-environnement tumoral: rôle de la protéase ...

Materials and MethodsEthics StatementAll subjects gave their informed written consent to participate to the study, andinvestigations were performed in accordance with the declaration of Helsinki asrevised in 2000 (http://www.wma.net/e/policy/b3.htm).Cells and cell cultureCell lines were cultured in DMEM (Invitrogen) supplemented with 10% fetal calfserum (FCS). Differentiation was induced by incubating 3T3-F442A confluent cells indifferentiation medium (DMEM supplemented with 10% FCS and 50 nM insulin) asdescribed (Prawitt et al., 2008). Differentiation was induced by incubating 3T3-L1confluent cells in differentiation medium (DMEM supplemented with 10% FCS and10µg/ml insulin, 250 µM isobutylmethylxanthine, 1 µM rosiglitazone, 1 µMdexamethasone).RNA extraction and analysisTotal RNA was extracted using the Reasy minikit (QIAGEN Sciences, Maryland)according to the manufacturer's instructions. Reverse transcription of total RNA wasperformed at 37°C using Moloney murine leukemia virus reverse transcriptaseenzyme (Invitrogen, Carlsbad, CA) and random hexanucleotide primers (Promega,Madison, WI). Quantitative PCR was carried out by real-time PCR using aLightCycler and the DNA double-strand-specific SYBR green I dye for detection(Roche, Basel, Switzerland). Results were normalized to RS9 levels. Sequences ofprimers are:12