Complete dulbecco s modified eagle s medium

Human HN6 and HN12 cell lines were a gift from A. Yeudall in 2016 and maintained in our laboratory (43 (link)). UM-SCC1 (SCC1) cells were obtained courtesy of T. E. Carey (University of Michigan, Ann Arbor, MI). Mouse oral squamous cell carcinoma MOC2 cells were obtained from Kerafast (Boston, MA). All cells were used for experiments before passage 10 and cultured in complete Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum (Biological Industries), 2 mM l-glutamine (Biological Industries), and 1% penicillin-streptomycin (Gibco, USA) at 37°C in a humidified incubator supplied with 5% CO₂. All cell lines were not genetically authenticated but were routinely screened for mycoplasma contamination by MycoAlert Mycoplasma Detection Kit (Lonza).

293T cells (ATCC, Manassas, VA, USA) were cultured in complete Dulbecco's modified Eagle's medium (Life Technologies) supplemented with penicillin (50 U ml⁻¹), streptomycin (50 μg ml⁻¹), 2 mM of L-glutamine (Life Technologies) and 10% heat-inactivated FCS (Sigma, St Louis, MO, USA), then cotransfected with 10 μg of pNL4-3 (NIH AIDS Research and Reference Reagent Program) and 5 μg CD44 plasmid (to generate HIV_CD44), or with empty pcDNA3.1/V5-His TOPO TA Expression vector (to make HIV_MOCK) using FuGENE HD Transfection Reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. Three days later, the supernatant was collected and HIV production was assessed using an HIV-1 p24 ELISA assay (PerkinElmer, Waltham, MA, USA).

The present work did not include any materials obtained directly from human participants and only used MHCC97-H cells purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The usage of the cell lines was permitted by the ethics committee of the Fifth Medical Center, General Hospital of the Chinese PLA (previously named the Beijing 302 Hospital). All experiments were performed according to the Declaration of Helsinki (World Health Organization).45 (link)
For proliferation analysis, cells were seeded in 96-well plates (5 × 10³ cells per well) (Corning, NY, USA). Cells were cultured in DMEM (complete Dulbecco’s modified Eagle’s medium, Invitrogen, USA) with 10% FBS (fetal bovine serum, Invitrogen, USA) at 37°C with 5% CO₂ for 24 h.41 (link) Treat the MHCC97-H cells with indicated concentration (10.0 μmol/L, 3.0 μmol/L, 1.0 μmol/L, 0.3 μmol/L, 0.1 μmol/L, 0.03 μmol/L or 0.01 μmol/L) of molecular targeting agents for 48 h in MTT experiments.49 (link),50 (link) The relative survival-cell number was reflected by OD 490 nm and the inhibitory rates of molecular targeting agents on MHCC97-H cells were calculated as (control group’s OD 490 nm – administration group’s OD 490 nm)/(control group’s OD 490 nm) × 100%. The IC₅₀ values of molecular targeting agents on MHCC97-H cells were calculated by inhibitory rates.51 (link),52 (link)

As previously described, normal esophageal epithelial cells (NEECs) were constructed from fresh samples of adjacent relative normal esophageal tissue, 5 cm far away from the cancerous section.25 (link) Professors SW Tsao and G Srivastava (University of Hong Kong) kindly provided us with some ESCC cell lines such as Eca109 and EC18. The remaining cell lines used in this study, including KYSE-520, KYSE-410, KYSE-30, KYSE-140, and KYSE-510, were all purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany), German Resource Center for Biological Material.26 (link) Complete Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) was used to culture the cells. The use of all cell lines was approved by the Yantai Yuhuangding Hospital Institution Review Board.