Cell proliferation assay

Cells were stably transfected with different plasmids and then plated in 96-well plate. Cell proliferation assay were performed using Cell Counting Kit-8 purchased from Solarbio Life Science as manufacture’s instruction. Crystal violet staining assay was carried out by plating 1 × 10⁴ cells in six-well plate. Then cells were grown in with or without E₂ for a week and subjected to crystal violet staining as previously described.³⁷ After staining, wells were washed three times with phosphate-buffered saline and destained with acetic acid, and the absorbance of the crystal violet solution was measured at 590 nm. For cell cycle assay, cells were transfected with the appropriate plasmids and subjected to Flow Cytometry assay with PI staining. Data were collected by FACSCalibur (BD Biosciences). Results were analyzed using ModFit software (BD Biosciences).