Scramble sirna

MCF-7 cells were maintained at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 1% nonessential amino acids and 2% of penicillin/streptomycine. The cell line has been authenticated by Eurofins. Prior to treatment with ligands, cells were grown for 48 h in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest), in order to remove steroid hormones or in serum-free medium for IGF-1 treatment. The cells were then treated for different times with E₂ (Sigma) 10^–8 M or IGF-1 (4×10^–5 µg/µl) from Peprotech. When stated, cells were treated with the PRMT1 inhibitor MS023 (Tocris Bioscience).
For knockdown experiments, specific siRNAs or scramble siRNA (Eurogentec) (50 nM) were transfected into MCF-7 cells using the lipofectamine 2000 reagent (Invitrogen). The targeted sequences are given in Supplementary Table 1. After 72 h of transfection, proteins were analyzed.
For overexpression experiments, pSG5-Flag-tagged vectors were transfected into MCF-7 cells using Jetprime reagent (Ozyme) according to the manufacturer’s protocol. Thirty hours after transfection, cells were collected and analyzed.

ERα+ cells lines (MCF7, Cama1) and ERα– cell lines (MDA-MB-231, Skbr3 and HBL100) were obtained from the ATCC. All of the cell lines were maintained at 37 °C in the appropriate medium supplemented with 10% foetal bovine serum, 1% non-essential amino acid and 2% penicillin/streptomycin. All cell lines were regularly tested for mycoplasma contamination. We also used frozen tumours from human breast cancer-derived xenografts (PDX) from Dr. Marangoni. HBCc-12A- (ERα–) and HBCx-3-expressing (ERα+ ) cell lines were established from the human breast cancer xenografts as described by Marangoni et al.^{26 (link)}For knockdown experiments, furin-specific siRNAs from ThermoFisher Scientific (ambion s9988 and s9987) siRNA furin 1: sens: 5′-GGGCCUUCAUGACAACUCATT-3′; anti sens: UGAGUUGUCAUGAAGGCCCAG; siRNA furin 2: sens: GGUGGAAAAUGGACUGGCUTT; anti sens: AGCCAGUCCAUUUUCCACCTT; the scramble siRNA (Eurogentec) (20 nM) was transfected into MCF‐7 and MDA-MB-231 cells using the lipofectamine 2000 reagent according to the manufacturer’s instructions (Invitrogen).