SK-N-BE and MCF-7 cell lines (ATCC, LGC Standards S.r.l., Milan, Italy) were used at passage 4–8 and were grown in air containing 5% CO2 in phenol red free, RPMI-1640 or DMEM medium, respectively, containing 10% (v/v) charcoal-stripped fetal bovine serum, L-glutamine (2.0 mM), Pen-Strep solution (penicillin 100 U/ml and streptomycin (100 mg/ml) as previously described [8 ,24 (link)] (Control Medium). Nutrient deprivation condition was obtained by culturing cells in amino acid and serum free, Earle’s Balanced Salt Solution (EBSS, Sigma Aldrich) containing 1 g/L of D-glucose for the indicated times. Stable ERα-transfected HEK-293 (ERα-HEK-293) cell lines were routinely grown in media containing G418 50 mM [25 (link)]. Cell line authentication were periodically performed by amplification of multiple STR loci by BMR Genomics srl (Padua, Italy). Cells were simultaneously treated with the vehicle used to dissolve all drugs (ethanol/PBS 1:10, v/v), and/or E2 (1 or 10 nM), and/or Bafalomycin-A1 (Baf-A1, 100 nM). When indicated, E2 or Baf-A1 pretreatment were performed adding the compounds 1 h before. For nutrient deprivation, MCF-7, SK-N-BE or ERα-HEK-293, were cultured as above reported, washed 3 times with PBS then cultured in EBSS for the indicated time points.
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