Fadu htb 43

HNEC were obtained by nasal brushings of 13 healthy, non-smoking, volunteers (5 males, 8 females, age 26–51) as described by O'Brien et al. [21] (link). Cells were maintained in collagen-coated tissue culture flasks in complete medium containing keratinocyte serum-free medium (KSFM) (Invitrogen, Paisley, UK) supplemented with 0.05 mg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, NY, USA). In the experiments cells from passages 2–5 were used and they were all positive for EpCAM (>90%), an adhesion molecule specific for epithelial cells [22] .
Human pharyngeal epithelial cell lines Detroit-562 (CCL-138) and FaDu (HTB-43) (ATCC, Manassa, USA) was cultured in complete medium containing minimum essential medium (MEM) with Earl's salts and 2 mM L-glutamine (Gibco), supplemented with 10 % FBS (PAN Biotech, Aidenbach, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). The medium for Detroit-562 also contained 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, USA), 0.1 mM non-essential amino acids, 50 µg/ml gentamicin and 0.25 µg/ml fungizone (Gibco). All cells were cultured at 37°C in a humidified 5% CO₂ air atmosphere.

The normal oral epithelial cell line OKF6 was purchased from the Harvard Skin Disease Research Centre (HSDRC, Boston MA). The oral cancer cell lines SCC9 (CRL-1629), SCC15 (CRL-1623), SCC25 (CRL-1628), and CAL27 (CRL-2095), A253 (HTB-41) and FaDu (HTB-43) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cell lines were authenticated and validated by short tandem repeat (STR) profiling and tested negative for mycoplasma contamination^{33 (link)}. OKF6 and SCC25 were cultured in keratinocyte serum-free medium (K-SFM, Gibco^TM) supplemented with growth factors (25 μg/mL BPE, 0.2 ng/mL EGF and 0.3 mM CaCl₂) and 1% penicillin-streptomycin (P/S). SCC9, SCC15, and CAL27 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco^TM) with 10% foetal bovine serum (FBS) and 1% P/S. All cell lines were cultured at 37 °C in a 5% CO₂ humidified incubator and maintained at less than 25 passages. To activate the PI3K signalling, cells were grown in normal media and treated with EGF (100 ng/mL) for 30 minutes. To inhibit the PI3K signalling, cells were grown in normal media and treated with 100 nM BEZ235 (HY-50673, MedChemExpress) for 6 hours. The media was removed, and cells were washed with phosphate-buffered saline (PBS), then lysed in RIPA buffer for western blot analyses.