Cell Line Cultivation and Characterization

The cutaneous squamous cell carcinoma A431(CRL–1555) and grade IV lung squamous cell carcinoma SW900 (HTB–59) cell lines, were purchased from American Type Culture Collection (ATCC) and grown according to the manufacturer’s suggested conditions in complete medium (1× DMEM (ATCC, 30–2002) for A431cells or 1× ATCC-formulated Leibovitz’s L–15 Medium, Catalog No. 30–2008 medium (ATCC, 30–2001) for SW900 cells) supplemented with 10% FBS and 1× penicillin-streptomycin grown at 37°C in 5% CO2. The cutaneous squamous cell carcinoma SCC13 cell line [81 (link), 82 (link)], were obtained from Harvard Skin Disease Research Center (HSDRC) and grown using the human keratinocyte culture methods provided by the HSDRC. The cutaneous squamous cell carcinoma COLO16 cell line [83 (link)] were grown in DMEM/Ham’s F12 50/50 media supplemented with cholera toxin (Sigma, C0852), insulin (Sigma, I5500-50MG), epidermal growth factor (Serotec/Bio-Rad, EGF-1), hydrocortisone (Sigma, H4881-1G), liothyronine (Sigma, T6397-100MG) and apo-transferrin (Sigma, T2252-100MG). All cell lines were verified human origin and free from pathogens and mycoplasma. Mycoplasma infection monitoring was performed using MycoAlert Detection Kit (Lonza) and only mycoplasma-free cultures were used.