Fig 1: Cell proliferation, cell cycle, and apoptosis are affected upon treatment of ERα+ breast cancer cells with ERβ-specific agonists, OSU-ERb-12, and LY500307. MCF7 and T47D cells (0.5 × 106) were seeded on 100-mm dishes in phenol red free DMEM containing charcoal-stripped FBS and treated with the drugs as indicated. (A) A representative diagram of the cell proliferation profile in drug-treated cells. Cells were treated with DMSO (control), FAS (fulvestrant; negative control), OSU-ERb-12, or LY500307 for 72 h, harvested, and stained following protocol for the Click-iT EdU Alexa Fluor 647 Kit (Invitrogen C10424). Cell proliferation was analyzed via flow cytometry on a BD FACSCalibur Flow Cytometer. Each assay was performed in triplicate and repeated twice. Data were plotted as mean ± SD (*p < 0.05, **p <0.01). (B) A representative diagram depicting the cell-cycle profile in drug-treated cells. Cells treated with DMSO (control), OSU-ERb-12, or LY500307 for 72 h at the indicated concentrations were harvested on ice, fixed, washed, and incubated with propidium iodide and RNase A followed by cell-cycle analysis on a flow cytometer. Each assay was performed in triplicate and repeated twice. Data were plotted as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). (C) A representative diagram depicting the apoptosis profile in drug-treated cells. Cells treated with DMSO (control), OSU-ERb-12, or LY500307 for 48 h at the indicated concentrations were harvested on ice, washed, and processed according to the manufacturer’s protocol (TUNEL Assay Kit-BrdU-Red; Abcam) followed by analysis on a BD FACSCalibur Flow Cytometer. Each experiment was repeated twice. Data presented are mean ± SD (*p < 0.05, **p < 0.01). In all assays, results shown are pooled averages across biological repeats.