ESR1 and breast cancer: To investigate E2-induced DNA damage in vitro, we treated an ERα-positive breast cancer cell line, MCF-7, with or without estradiol (E2), the most active form of estrogen, for 2 h and counted the number of the signals of phosphorylated-histone H2AX (gamma-H2AX, gH2AX), a marker of DNA double-strand breaks (Figure S1A).