Mccoy s medium

Human malignant melanoma cell lines (part of NCI-60 panel of human cancer cells) - SK-MEL-5, SK-MEL-28, MALME-3M, MDA-MB-435S and normal human skin fibroblast cells MALME-3 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). SK-MEL-5 and SK-MEL-28 were grown and maintained in EMEM media (ATCC), while MALME-3M and MDA-MB-435S were grown in IMDM (ATCC) and RPMI-1640 (ATCC) respectively, as indicated by ATCC protocols. Complete growth media was supplemented with 10% fetal bovine serum (FBS; ATCC) and 1X antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA). MALME-3 cells were grown in McCoy's medium (ATCC) supplemented with 15% FBS and 1X antibiotic-antimycotic. Cells were grown at 37C / 5% CO₂ in a humidified incubator. Half the media was replaced every 48 hr. No hGH was present in the media or added externally unless specifically mentioned. For hGH treatment, 16 hr. after seeding (or 24 hr. post-transfection), the cells were serum-starved for 2 hr. in serum free growth media and hGH (PBS as control) was added at the mentioned concentrations (5, 50, 150 ng/mL). Cells were subsequently incubated for 24 hr. before RNA extraction. Recombinant hGH was purchased from Antibodies Online (Atlanta, GA).

The melanoma cell line Mel526 was a gift from Prof. Magnus Essand (Uppsala University, Uppsala, Sweden) and was cultured in RPMI 1640 supplemented with l-glutamine, 10% fetal calf serum (FCS), and 1% penicillin/streptomycin (PeSt). Two human pancreatic cell lines (MiaPaCa2 and Panc01) were gifts from Dr. Rainer Heuchel (Karolinska Institute, Stockholm, Sweden) and cultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. The ovarian cancer cell line SKOV3 was purchased from ATCC (Manassas, VA, USA) and cultured in McCoy’s medium supplemented with 10% FCS and 1% penicillin/streptomycin. All media and supplements were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The cells were incubated at 37°C in 5% CO₂ and humidity. To investigate the surface expression of CD40L and 4-1BBL, Mel526 cells were infected with an MOI of 10 fluorescence-forming units (FFU)/cell of LOAd703, LOAd700, or LOAd(−) for 2 h and incubated for 48 h at 37°C prior to cell harvest and staining for flow cytometry.