Airway epithelial cell growth medium

Five HNSCC tumor specimens obtained just after surgery were provided through collaboration with the Centre Antoine Lacassagne (Pr A. Bozec). Viable tumor sections (250 μm, tumor samples were confirmed by a pathologist (Dr J. Boyer)) were obtained with a vibratome HM650V machine. They were seeded in Airway Epithelial Cell Growth Medium (PromoCell) supplemented with NormocureTM (Invivogen) and treated with a range of cabozantinib or cisplatin concentrations. Tumor sections were then paraffin-embedded and analyzed using HES for the quantification of necrotic areas and TUNEL staining (In Situ Cell Death Detection Kit, sigma) for the quantification of dead cells. Tumor sections were lysed and the ATP concentration evaluated (ATP Bioluminescence Assay Kit HSII, Roche), normalized to the protein concentration and represented as a read-out of viability (see Figure 7 and Table S2).

Experiments in this study were conducted using BEAS-2B cells (RRID: CVCL_0168), A549 cells (RRID: CVCL_0023), and HNEpC. These are respiratory epithelial cell types widely used as in vitro cell culture models. BEAS-2B and A549 cell lines were purchased from Duke Cell Culture Facility (Source: ATCC, Manassas, VA) and HNEpC from PromoCell GmbH (Heidelberg, Germany). BEAS-2B cells were cultured in BEGM Bronchial Epithelial Cell Growth Medium BulletKit (Lonza, Walkersville, IL) containing BEGM basal medium and SingleQuots supplements and growth factors. Cells were plated onto flasks and plates coated with a mixture of fibronectin (0.01 mg/mL; Sigma-Aldrich, Saint Louis, MO), bovine collagen type I (0.03 mg/mL; Sigma-Aldrich) and bovine serum albumin (0.01 mg/mL; Sigma-Aldrich) dissolved in cell culture medium and overnight incubated at 37°C in a 5% CO₂ and 95% humid atmosphere. A549 cells were cultured in F-12K medium supplemented with 10% fetal bovine serum and 100 units/mL penicillin and 0.1 mg/mL streptomycin. HNEpC were cultured in Airway Epithelial Cell Growth Medium (PromoCell) that contained growth supplements, growth factors and antibiotic–antimycotic mix (10 000 units/mL of penicillin, 10 000 µg/mL of streptomycin, and 25 µg/mL of Amphotericin B; ThermoFisher Scientific, Waltham, MA).

Tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, an ER stress inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary human nasal epithelial cells (HNEpCs) and airway epithelial cell growth medium were purchased from PromoCell (Heidelberg, Germany). RPMI 1640 medium and Trizol were from Invitrogen (Carlsbad, CA, USA). EZ-Cytox Cell Viability Assay kits were purchased from Daeil Laboratories (Seoul, Korea). Reverse transcriptase-polymerase chain reaction (RT-PCR) kits were from Applied Biosystems (Foster City, CA, USA). Real-time PCR kits were obtained from Roche Applied Science (Mannheim, Germany). Primary and secondary antibodies for MUC5AC and MUC5B (used immunoassay) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For Western blot analysis, primary and secondary antibodies of XBP-1, CCAAT-enhancer-binding protein homologous protein (CHOP), ATF6 and β-actin were purchased from Abcam (Cambridge, MA, USA). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT, USA). OPTI-MEM I Reduced Serum Medium, Lipofectamine 2000, predesigned small interfering RNAs (siRNAs) targeting XBP-1, CHOP, and ATF6, and control siRNA were purchased from Thermo Fisher Scientific (Lafayette, CO, USA). This study was approved by the Institutional Review Board of Yeungnam University Medical Center (IRB No. YUMC 2015-06-058).