Ishikawa Cell Culture and UPA Treatment

Ishikawa cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco), and maintained at 37 °C in 5% CO₂. UPA in powder form was obtained from ASKA Pharmaceutical (Tokyo, Japan) and used as a chemical reagent. It was dissolved in 0.1% dimethyl sulfoxide, and an appropriate concentration thereof (0, 10, or 40 μM) was applied based on the previous reports [15 , 16 (link)]. The estrogen receptor (ER) antagonist ICI 182,720 (hereinafter referred to as “ICI”) was also dissolved in 0.1% dimethyl sulfoxide, and its concentration (1 μM) was determined based on previous reports [17 (link), 18 (link)]. DMEM/F-12, without phenol red (Thermo Fisher Scientific, Waltham, MA, USA) was used with the addition of ICI because phenol red has a weak estrogenic effect [19 (link)]. Mouse monoclonal antibody anti-β-actin (sc-47778), anti-p53 (sc-126), anti-Bax (sc-20067), and anti-Bcl-2 (sc-7382) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), whereas anti-cleaved PARP (cat. no. 5625) was obtained from Cell Signaling Technology (Danvers, MA, USA).