Bovine pituitary extract

The human endocervical epithelial End1/E6E7 cell line, the human cervical cancer HeLa, C33A and SiHa cell lines, and the cervical squamous cell carcinoma Ca-Ski cell line were purchased from BioVector NTCC, Inc. End1/E6E7 cells were cultured in keratinocyte serum-free medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 50 µg/ml bovine pituitary extract (Absin Bioscience, Inc.), 0.1 ng/ml recombinant epidermal growth factor (Absin Bioscience, Inc.), 0.4 mmol/l CaCl₂ and 1% antibiotics (penicillin, 100 U/ml; streptomycin, 100 mg/ml). HeLa and CaSki cervical cancer cell lines were cultured in RPMI-1640 (Procell Life Science & Technology Co., Ltd.) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin. C33A and SiHa cells were cultured in DMEM (MilliporeSigma) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin. All cells were cultured under the conditions of 37°C with 5% CO₂.

Authenticated immortalized HMGECs (a generous gift from David Sullivan, Schepens Eye Research Institute, Boston, MA, USA) were cultured in keratinocyte serum-free medium (KSFM; Invitrogen-Gibco, Grand Island, NY, USA) supplemented with epidermal growth factor (EGF, 5 ng/mL; Invitrogen-Gibco) and bovine pituitary extract (50 µg/mL; Absin, Shanghai, China) as previously described.³ (link) At 80% confluency, the cell medium was changed by Dulbecco's modified Eagle's medium and Ham's F12 (DMEM-F12; Invitrogen-Gibco) supplemented with EGF (10 ng/mL) and 10% fetal bovine serum (Invitrogen-Gibco) for cellular differentiation. The HMGECs pretreated with MLT (Sigma-Aldrich, St. Louis, MO, USA) or isochoric dimethyl sulfoxide (DMSO; Sigma-Aldrich) for 48 hours were characterized as the MLT group and the control group, respectively, while those that subsequently received 10 µg/mL LPS (Escherichiacoli, strain 0127:B8, Sigma-Aldrich) were defined as LPS + MLT group and LPS group, respectively. The dose of LPS was used as previously described.³³ (link)

Normal prostatic epithelial RWPE-1 and HPr-1 cells (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) were cultured in complete keratinocyte serum-free medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), supplemented with 1% penicillin/streptomycin/amphotericin B, 50 μg/ml bovine pituitary extract (Absin Bioscience, Inc., Shanghai, China) and 5 ng/ml epidermal growth factor (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were incubated at 37°C in an atmosphere containing 5% CO₂. When cells reached 70-80% confluence they were washed with PBS and detached using 0.25% trypsin/0.2% EDTA. Cells were viewed under a light microscope (magnification, ×200; Eclipse T1, Nikon Corporation, Tokyo, Japan), and subsequently suspended at a concentration of 1×10⁶ cells/ml.