Charcoal stripped serum

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.

Three breast cancer cell lines expressing ERα, namely MCF7, T47D, and ZR75-1, were used in this study. Transient transfections were carried out in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest) in order to remove steroid hormones (steroid depletion). Cells were transfected with 20 nM siRNA of, respectively, control siRNA (5 nmoles, Eurogentec), two different siRNA targeting human MEN1 transcript (siMEN1 hs1 (HSS106462) and hs2 (HSS181079), ThermoFisher Sci.), siRNA targeting human MLL1 (SiKMT2A: siRNA 107,890 ThermoFisher Sci.), siRNA targeting human MLL2 (SiKMT2B: siRNA s18833 ThermoFisher Sci.) using Jetprime® transfection reagent (Polyplus) for 72 h according to the manufacturer’s instructions. Inhibition of the menin–MLL interaction was achieved by MI503 (Active Biochem) at different concentrations. Prior to performing treatment with E2 and MI503, cells were grown in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest) in order to remove steroid hormones (steroid depletion). Cells were then treated for 3 h with E₂ (Sigma) 10⁻⁸ M and MI503 for 48 h. The treatment was repeated after 24 h due to the degradation of the inhibitor with time. Please also see Additional file 2—Supplemental Materials & Methods.

T47D (ATCC) cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 2% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA), and insulin (10 µg/mL). They were grown in a humidified atmosphere with 5% CO₂ at 37 °C, authenticated by Eurofins and tested for Mycoplasma infection by the MycoAlert Mycoplasma Detection Assay (Lonza, Rockland, ME, USA).
Prior to experiments, T47D cells were grown in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest, Nuaillé, France). Then, 48 h later, medium was replaced by fresh serum-free medium for 24 h to starve the cells. When indicated, the cells were treated with the MS 023 Type I PRMT inhibitor (Tocris, Bristol, UK) for 48 h at 60 nM (or DMSO vehicle) or with the synthetic progestin R5020 (Perkin Elmer, Waltham, MA, USA) for 6 h.