Recombinant epidermal growth factor

The VK2/E6E7 vaginal epithelial cell line (ATCC® CRL-2616), obtained from the American Type Culture Collection (ATCC; Rock ville, MD, USA) is an epithelial cell line derived from the vaginal mucosa of a healthy premenopausal female undergoing vaginal repair surgery, that was subsequently immortalized with human papillomavirus 16/E6E7. VK2 cells were cultured in keratinocyte-serum free medium (K-SFM, Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 µg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA), 100 U/mL penicillin (Life Technologies, Grand Island, NY, USA), 100 µg/mL streptomycin (Life Technologies) and 0.4 mM CaCl₂ at 37 °C with 5% CO₂ and a high humidity environment. A subculture of the cells was performed every 3–4 days.

SCC-9, SCC-25 and CAL 27 cells were purchased from the American Type Culture Collection (ATCC, U.S.A.), and the human HNSCC cell lines HN4, HN6 and HN30 were kindly provided by the University of Maryland Dental School, U.S.A. SCC-4, SCC-9 and SCC-25 cells were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (GIBCO-BRL, U.S.A.) supplemented with 10% fetal bovine serum (GIBCO-BRL, U.S.A.) and 1% penicillin and streptomycin at 37°C, 5% CO₂ in a humidified atmosphere while other cells were maintained in DMEM with the same additives. Furthermore, normal oral epithelial cells which were obtained from primary culture were cultured in keratinocyte serum-free medium (GIBCO-BRL, U.S.A.) containing 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, U.S.A.).

HEK293 (ATCC no. CRL-1573) human embryonic kidney cell, MRC5 (ATTCC no. CCL-171) human non-cancerous fibroblasts, HBEC (ATTCC no. CRL-4051) human non-cancerous epithelial cells, A549 (ATCC no. CCL-185) human lung carcinoma, H1299 (ATCC no. CRL-5803) metastatic human lung carcinoma of the lymph node, H441 (ATCC no. HTB-174) human lung papillary adenocarcinoma, MCF-7 (ATCC no. HTB-22) human breast adenocarcinomas, and Saos-2 human bone osteosarcoma (ATTCC no. HTB-85) cell lines were from the American Type Culture Collection (Rockville, MD, USA). A549, MCF-7, MRC5, and HEK293 cells were maintained in DMEM. H1299 and H441 cells were cultured in RPMI 1640 medium. Saos-2 cells were maintained in McCoy’s 5A media. HBEC cells were maintained in keratinocyte serum-free medium containing bovine pituitary extract and recombinant epidermal growth factor (Invitrogen, Waltham, MA, USA). All cell culture media was supplemented with 10% fetal bovine serum, 5% l-glutamine, and 5% penicillin/streptomycin (100 U/mL) unless otherwise indicated. All cells were cultured and maintained in humidified 5% CO₂ incubators at 37 °C. All other cell culture reagents were obtained from VWR (VWR, Radnor, PA, USA) unless otherwise indicated.

Vk2 (E6/E7) cells (CRL-2616) were obtained from American Type Culture Collection (Rockville, MD). Cells were grown in keratinocyte-serum free medium (K-SFM) containing 5 ng/ml recombinant epidermal growth factor and 50 µg/ml bovine pituitary extract all purchased from Invitrogen Corporation (Grand Island, NY). A maximum tolerated dose of N9 was determined by serially diluting the microbicide from 1% to 0.001% in cell culture medium and each dilution added to individual wells of an 80–90% confluent monolayer of Vk2 Cells in 96-well plates. Cell viability was performed using CellTiter-Glo.

HK1, HK1-EBV, C666-1 and HeLa cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% L-glutamine (Life Technologies). The NP69 cells were cultured in keratinocyte serum-free medium (KSFM) with bovine pituitary extracts and recombinant epidermal growth factor (Invitrogen). All of the cells were incubated in a humidified atmosphere with 5% CO₂ at 37°C.
Two Silencer Select validated siRNAs targeting PIN1 (siPin1 544 and siPin1 545) and universal negative control siRNA (Life Technologies) were transiently transfected into C666-1 using Lipofectamine^TM 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s protocols. Cells were collected at indicated time points for further analysis. MG132 (Calbiochem) was applied to HK1 and NP69 cells (at 10 and 15 μM and 5 and 10 μM, respectively) to study the proteasome degradation of PIN1. The PIN1 inhibitor Juglone (Calbiochem) was used to study the effect of PIN1 inhibition on C666-1 cells (at 2, 4, 6 and 8 μM). Treated cells were collected and stored at -80°C until use. To determine the IC50 of Juglone, the cells were seeded into 96-well plates and treated with different Juglone dosages (0.03–20 μM) for 24 hours. Cell viability was then determined by a WST-1 assay.

Salmonella strains (Table 1) were preserved at −80°C in 15% (vol/vol) glycerol. Salmonella were grown on brain heart infusion (BHI) (Becton Dickinson, Sparks, MD) agar plates, which were incubated at 37°C. For in vitro infections, single colonies of Salmonella isolates grown on BHI agar plates were inoculated into 5-ml aliquots of LB (pH 8; 0.3 M NaCl), followed by incubation under static conditions at 37°C for 12 to 14 h. These cultures were subsequently subcultured 1:100 into fresh aliquots of LB (pH 8; 0.3 M NaCl), followed by incubation at 37°C under static conditions, until mid-log phase (optical density at 600 nm [OD₆₀₀] of 0.4 to 0.5).
Human intestinal epithelial cells (HIEC-6 cells; ATCC), derived from human fetal ileum (51 (link)), were grown in Opti-MEM medium supplemented with recombinant epidermal growth factor (10 ng/ml; Gibco-Invitrogen, Carlsbad, CA) and 10% (vol/vol) fetal bovine serum (FBS) (Gibco-Invitrogen) and were incubated at 37°C with 5% CO₂. HIEC-6 cell supernatants were routinely tested for Mycoplasma infection using the VenorGEM Mycoplasma detection kit (Sigma-Aldrich, St. Louis, MO). For Salmonella enterica serotype Javiana infections, HIEC-6 cells were grown in 6-well or 24-well plates (Corning, Corning, NY). Cells were seeded into 6-well (2 × 10⁵ cells per well) or 24-well (1 × 10⁵ cells) plates 48 h (+4) h before infection.