Hep 2 cell line

The human Hep-2 cell line (CRL-9609), and the human lung adenocarcinoma A549 epithelial cell line (CCL-185) were purchased from the ATCC (Manassas, VA, USA). Cells were grown in DMEM (high glucose, Gibco, USA) supplemented with penicillin/streptomycin and 10% heat-inactivated FBS, in a humidified 5% CO2 atmosphere at 37°C. Hep-2 cells were initially used to spread the viruses in a better way or use infection in place of spread. Similarly, after 3 days, the lesions of each pore were observed. When more than 50% of the cells were diseased, the pore was considered to be infected with RSV (or syncytia). The result was calculated by Reed-Muench method. When the virus infects the cells, DMEM containing 2% FBS is used, and the virus that is not bound to the virus is washed twice with PBS.

HEp-2 cell line was obtained from ATCC (Manassas, VA, USA). HEp-2 cells were cultured in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (ThermoFisher Scientific) and penicillin–streptomycin-glutamine mixture (ThermoFisher Scientific) at 37 °C in 75 cm² tissue culture flasks. About 100 million cells were harvested and used for protein extraction. Harvested cells were suspended in 10 mL of 50 mM phosphate buffer (pH 7.4) containing the Roche Complete Mini protease inhibitor cocktail (Sigma Aldrich). Cells were homogenized on ice with a microprobe sonicator until the turbid mixture became nearly clear with no visible cells left. The homogenate was centrifuged at 10,000g at 4 °C for 20 min, and the supernatant was collected as the total protein extract. Protein concentrations were measured with the Bio-Rad RC DC protein assay.

Vero cell line (African green monkey kidney epithelial cells, ATCC CCL-81), HeLa cell line (ATCC, CCL-2), HEp-2 cell line (ATCC, CCL-23) and human neuroblastoma SK-N-SH cells (ATCC, HTB-11) were purchased from ATCC (Manassas, VA) or from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Mouse embryo fibroblast (MEF) cells (isolated from C57BL/6J mouse embryos) were prepared in the lab following published protocol [64 (link)]. The cells were cultured in DMEM (high glucose) supplemented with 10% heat-inactivated fetal bovine serum (FBS), sodium pyruvate and non-essential amino acids (Life Technologies, Carlsbad, CA) in a humidified incubator at 37°C with 5% CO₂. HSV-1 strain F and HSV-2 strain G were propagated and titrated on Vero cells.

Two cell lines derived from normal or neoplastic tissues were used in the present study, the HUVEC cell line (CRL-2873; AMERICAN TYPE CULTURE COLLECTION/ATCC, Manassas, VA, USA), which was obtained from normal human umbilical vein/vascular endothelium, and the HEp-2 cell line (CCL-23; ATCC), which was originally established from an epidermoid carcinoma of the larynx. HUVEC was selected for the present study because it is one of the most popular cell lines used for many studies, including for experiments on inflammation^{91 (link)}. Hep-2 cells were used as a cancer cell model with similar epithelial origin of HUVEC cells⁹² .
HUVEC and HEp-2 cells were cultured in MEM-Earle medium (CULTILAB) supplemented with 10% FBS, 10 mM nonessential amino acids (GIBCO, Carlsbad, CA, USA), 2 mM l-glutamine, 1 mM sodium pyruvate (SIGMA ALDRICH) and 0.1% antibiotic/antimycotic (SIGMA ALDRICH), in a humidified atmosphere with 5% CO₂ at 37 °C.
To investigate whether piplartine in the presence of annexin A1 plays a role in inflammatory and neoplastic processes, HUVECs and HEp-2 cells were treated with the peptide Ac_2-26, piplartine, both Ac_2-26 and piplartine, or LPS.

The HEP-2 cell line was purchased from ATCC, Rockville, MD, USA. The cells were allowed to grow in DMEM with heat inactivated FBS (10%). HEPES buffer, penicillin (100 U/ml), streptomycin (100 g/ml), l-glutamine (1%), and gentamycin (50 g/ml), were added to cells, then they were kept in a humid environment at 5% CO₂ and 37 °C.