Cell culture conditions and compounds

Human esophageal cancer cell lines EC9706 and EC109, human gastric cancer cell line MGC-803, human prostate carcinoma cell line PC-3, and primary normal human liver cells (HL-7702) were purchased from China Center for Type Culture Collection (CCTCC, Shanghai, China). EC9706 cell line has been proven to be esophageal carcinoma of the fungating type, which is poorly-differentiated squamous cell carcinoma, and EC109 cell line is well-differentiated [19]. HEECs were obtained from Wuhan PriCells Biomedical Technology Co., Ltd. (Wuhan, China). All the cell lines were cultured in RPMI 1640 medium, containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere, containing 5% CO2. Pure oridonin was isolated from Isodon rubescens, and DS2 was synthesized in our laboratory. The chemical structures are shown in Figure ​Figure1A1A and were confirmed by NMR and IR data. Purities were determined by HPLC and were all above 98%. DS2 and oridonin were dissolved in dimethyl sulfoxide (DMSO), aliquoted, and stored at −80°C. The concentration of DMSO in culture medium was kept below 0.1% (v/v), a concentration known not to affect cell proliferation.