Flow Cytometry Analysis of Cell Treatment

Flow cytometry analysis was performed as previously described [11 (link),16 (link),39 (link)]. Briefly, MCF-7 and BT474 cells were seeded at a density of 1 × 10⁶ cells/plate and their media was changed two times in every two days. Following an overnight treatment with 4-OHT (10⁻⁶ M) (Sigma) and SEL (10⁻⁷ M) (Selleckchem) alone and in combination, cells were collected with 0.1% FBS containing 1× PBS, washed, resuspended at 1–2 × 10⁶ cells/mL and fixed with ice cold ethanol for 24 h. Next day, cells were washed with 1× PBS and incubated in 10 ng
RNAase for one hour at room temperature. Cells were stained with 0.25% Propidium Iodide (#10008351, Cayman Chemicals, Ann Arbor, MI, USA) for one hour and results were analyzed by BD™ LSR II Flow cytometry analyzer (BD Biosciences Inc., San Jose, CA, USA). Following analysis, results were analyzed by FCS Express 6 Flow Cytometry Software (DeNovo Software, https://www.denovosoftware.com/site/Flow-RUO-Overview.shtml) and all experiments were repeated three times.