Cell culture (G1E-ER4, MEL)

The murine erythroleukaemia cell line (MEL, gift from Stu Orkin, Harvard Medical School Boston, USA) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, #435847) supplemented with 10% (v/v) foetal calf serum (FCS) (Interpath, #SFBSF13) and 1% (v/v) penicillin/streptomycin/glutamine (PSG) solution (Life Technologies, #10378016)³⁸. G1E-ER4 is a subclone of GATA-1 null erythroblasts which stably expresses a GATA-1-ER (estradiol receptor ligand binding domain) fusion protein³². G1E-ER4 cells were cultured in Iscove’s MDM medium (Life Technologies, #12440-061) supplemented with 15% heat-inactivated foetal calf serum (Interpath, #SFBSF13), 1% PSG (Life Technologies, #10378016), 62 μl of 10% monothioglycerol (Sigma-Aldrich, #M6145), 0.6% home-made Kit-ligand conditioned medium, 50ul mouse erythropoietin (Biolegend, #587606) in 500 ml IMDM medium³⁹. Kit-ligand conditioned medium was made from CHO cells (gift from Stu Orkin, Harvard Medical School Boston, USA)³². Exponentially proliferating G1E-ER4 cells were induced with the addition of 4-hydroxytamoxifen (tamoxifen) (Sigma, #H7904) to a final concentration of 0.1 nmol for 24 h to restore GATA-1-ER fusion protein into cell nucleus³². G1E-ER4 cells incubated with ethanol (1 ul ethanol in 10 ml media) was used as mock (NC) control.