Cell Line Characterization and Treatment

HEC1A, AN3CA, and KLE cell lines were provided by Dr. Churl Ki Min (Ajou University, Suwon, South Korea), and the RL95-2 cell line was provided by Dr. Jong-Min Kim (Dong-A University, Pusan, Korea). The Ishikawa cell line was a gift from Dr. Hyung Sik Kim (Pusan National University, Pusan, Korea). The EM-E6/E7/hTERT cell line was originated by Mizumoto and colleagues [35 (link)] and obtained from Dr. Paul J. Goodfellow (Ohio State University, Columbus, OH). ECC-1 cells were purchased from the ATCC. All cell lines were maintained in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM /F12; Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin-streptomycin (Gibco) at 37°C in a humidified atmosphere of 5% CO₂. For E₂ treatment, cells were maintained in phenol red-free DMEM/F12 (Gibco) with 5% charcoal-stripped FBS for 1 day followed by serum-free medium for 24 h before the experiment. The E₂ and E₂-BSA (bovine serum albumin) were purchased from Sigma. E₂-BSA was prepared according to the protocol for removing free E₂ as previously described [36 (link)]. Cycloheximide (CHX) and N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) were purchased from Sigma.