Human transferrin

Ham's F12 medium, PBS, 7.5% BSA fraction V, and penicillin/streptomycin were purchased from Invitrogen (Burlington, ON, Canada). Fetal calf serum and Dulbecco’s Modified Eagle’s Medium (DMEM) were from Wisent Inc (St-Bruno, QC); PDGF-AA and bFGF from PeproTech (Rocky Hill, NJ). PD169316 was from EMD Chemicals (San Diego, CA). Poly-D-lysine, poly-L-ornithine, human transferrin, insulin, HEPES, Triton-X-100, DTT were from Sigma-Aldrich. Western blotting reagents from GE Healthcare Life Sciences (Baie d’Urfe, QC); A2B5 mouse monoclonal antibody from American Type Culture Collection; rabbit polyclonal Ki67 conjugated with FITC from Abcam (Toronto, ON); mouse monoclonal anti-p27kip1 (BD Biosciences, Mississauga, ON); rabbit polyclonal p57 (H-91) from Santa Cruz; rabbit monoclonal phopho-CDC2 (TYR15) from Cell Signaling Technology (Danvers, MA); HRP-, FITC-, or Texas Red-conjugated secondary antibodies from Southern Biotechnology, Jackson Immunoresearch Laboratories (Cedarlane, Hornby, ON), BIO-RAD Canada (Mississauga, ON) or Invitrogen (Burlington, ON); Hoechst nuclear stain from Molecular Probes Inc. (Eugene, OR). The O4 antibody was a gift (Sommer and Schachner 1981). All other reagents were from Fisher Scientific (Whitby, ON), or VWR (Mont-Royal, QC)

Panc-1, HEK-293 T, BxPC1 and MiaPaCa 2 cell lines were maintained in DMEM and L3.6pl were maintained in RPMI, each supplemented with 10 % (v/v) FCS, and 1 % (v/v) penicillin and streptomycin. Patient derived cell lines 5061, 5072 and 5167 were maintained in RPMI 1640 with Glutamax (Life Technologies) supplemented with 10 % of fetal calf serum (FCS), 200 IU/ml of penicillin-streptomycin, 0.1 mg/ml gentamycin (Biochrom), 50 nmol/ml of human transferrin (Sigma-Aldrich), 0.01 μg/ml of bovine insulin (Sigma-Aldrich), 0.01 μg/ml of recombinant human epidermal growth factor (Pepro Tech), and 0.01 μg/ml of human basic fibroblast growth factor (Pepro Tech).
Cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO₂. All cell lines were used at low passage number not exceeding 30 passages.

For stimulation with recombinant EGF (10 ng/ml) (GIBCO, Thermo Scientific, MA, USA), and/or erlotinib inhibition (conc. as indicated) up to 24 h, SCaBER, J82, and HT1376 cells were cultured in serum-free media, containing human transferrin and 1% DMSO (Sigma-Aldrich). For p-SCC cells standard conditions (see Supplementary Information) were used. Cellular proteins were extracted in RIPA lysis buffer containing phosphatase inhibitors and quantified using the Pierce^TM BCA protein assay (Thermo Scientific, MA, USA). RNA was extracted using the Nucleospin RNA Plus Kit.

The following reagents were used for general cell culture: heat-inactivated fetal bovine serum (HI-FBS), penicillin-streptomycin, TrypLE Express enzyme with phenol red (Gibco), Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich). Caco-2 BBe1 cells (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC) supplemented with 10% HI-FBS (v/v), 1% penicillin-streptomycin and 10 μg/ml human transferrin (Sigma). T84 cells (ATCC) were maintained in 10% DMEM/F-12 medium (Gibco) supplemented with 5% (v/v) HI-FBS and penicillin-streptomycin. Both cell lines were maintained at 37 °C, 5% carbon dioxide in a water-saturated environment and were not used past passage number 32. The Countess automated cell counter (Life Technologies) was used for cell counting.