Penicillin g

RPMI-1640 medium (#21875034) and fetal bovine serum (#10270106) were from Gibco, Life Technologies, Grand Isle, NY. Glutamine, penicillin G, and streptomycin sulfate were from Biological Industries, Beit-Haemek, Israel. FA-free medium (#R1145), dialyzed fetal bovine serum (#F0392), and VBT (# V1377) were from Sigma-Aldrich, St. Louis, MO, USA. NCZ (#sc-3518), LAN B (#sc-203318), Y27632 (#sc-281642), and CyMl (#sc-500804) were from Santa Cruz Biotechnology, Dallas, TX, USA. Blebb (#13013) was from Cayman Chemical, Ann Arbor, MI, USA. FA (#J62937) was from Alfa Aesar, Tewksbury, MA, USA.

Twelve well-characterized human adherent cancer cell lines from different solid tumor types were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA): MCF7 and MDA-MB-231 for breast adenocarcinoma; A375 for malignant melanoma; A-431 for epidermoid carcinoma; A549 and NCI-H460 for lung carcinoma; PC-3 for prostate adenocarcinoma; LNCaP for prostate carcinoma; SW480 and HT-29 for colorectal adenocarcinoma; U-87 MG and T98G for glioblastoma as well as primary normal bronchial/tracheal epithelial cells. Jurkat acute T cell leukemia cells were provided as a gift from Professor Yoram Reiter at the Technion, Israel Institute of Technology.
MCF-7, MDA-MB-231, A375, A-431, HT-29, U-87 MG and T98G cells were grown in high glucose DMEM (Sigma-Aldrich, D5796). A549, NCI-H460, PC-3, LNCaP, SW480 and Jurkat cells were grown in RPMI-1640 medium (Sigma-Aldrich, R8758) supplemented with 10% FBS (Biological Industries, 04-007-1A) and 100 units/ml of penicillin G and 100 μg/ml of streptomycin (Biological Industries, 03-031-1B). Primary normal cells were grown in serum-free conditions in Airway Epithelial Cell Basal Medium (ATCC, PCS 300-030) supplemented with the adequate cell growth kit (ATCC, PCS-300-040). All cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

The primary cultures of orbital fibroblasts were established according to our previous study [16 (link)]. Briefly, the orbital tissues were minced aseptically in phosphate-buffered saline (PBS, pH 7.3) and then incubated with a sterile solution containing 0.5% collagenase and dispase (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) for 24 hours in an incubator filled with an atmosphere of 5% CO₂ and kept at 37°C. The mixture of digested orbital tissues was pelleted by centrifugation at 1,000 ×g and then resuspended in Dulbecco's Modified Eagle's Medium (DMEM, purchased from Gibco Life Technologies, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS) and a cocktail of antibiotics (Biological Industries, Kibbutz Beit Haemek, Israel), which was composed of 100 U/mL penicillin G and 100 μg/mL streptomycin sulfate (Biological Industries, Kibbutz Beit Haemek, Israel). The cultured orbital fibroblasts were used between the 3rd and 5th passages and the cell cultures at the same passage number were used for the same set of experiments.

Four well-characterized human adherent epithelial cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA): Scc4 (ATCC^® CRL1624^TM), Scc9 (ATCC^® CRL1629^TM), Scc25 (ATCC^® CRL1628^TM), and Cal27 (ATCC^® CRL2095^TM). A human adherent fibroblast cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA): Hs895sk (ATCC^® CRL-7636^TM). Cells were grown in DMEM/F-12 (Biological Industries, Kibbutz Beit-Haemek, Israel, 01-170-1A), supplemented with 2.5 mM L-glutamine (Biological Industries, 03-020-1A), 100 units/mL of penicillin G, 100 μg/mL of streptomycin (Biological Industries, 03-031-1B), 0.5 mM sodium pyruvate (Biological Industries, 03-042-1B) 400 ng/mL hydrocortisone (Sigma-Aldrich, 50-23-7) and 10% fetal bovine serum (Biological Industries, 04-007-1A). Cells were seeded and incubated to 70–80% confluence before experiments commenced. All cells were maintained in a humidified atmosphere at 37 °C with 5% CO₂.