Bronchial epithelial growth kit

All cell lines were originally purchased from the American Type Culture Collection (ATCC, Manassas, VA). Human SCLC cell lines (H128, H146, H187, H69, DMS114 and DMS153) were provided by the laboratory of Dr. Taofeek Owonikoko [47 (link)]. H128, H187, H69, and H146 cells were grown in RPMI 1640 (Gibco) with 7.5% fetal bovine serum (FBS). DMS114 and DMS153 cells were grown in Waymouth’s medium (Gibco) with 5% FBS. HeLa, U2OS, HEK293T, and HCT116 cells were grown in DMEM (Gibco) with 7.5% FBS. BEAS-2B were cultured in DMEM/F-12 medium with 10% FBS. Primary small airway epithelial cells (HSAEC) were grown in Airway Basal Medium (ATCC PCS-300-030) with one Bronchial Epithelial Growth Kit (ATCC PCS-300-040) according to manufacturer’s instructions. All cell lines were grown at 37°C under humidified conditions with 5% CO₂ and 95% air.
Details of additional methodologies can be found in Supplementary Materials and Methods.

Cancer cell lines were cultured in Airway Epithelial Basal Media supplemented with Bronchial Epithelial growth kit from ATCC. Cells were grown in 75 cm² sterile polystyrene culture flasks to 80% confluency, trypsinized, and re-seeded in equal aliquots into 96-well plates. After 2 days and ~50% confluency, media was removed and replaced with DMSO or drug containing media. Cells were allowed to grow another 4 days (all other fast growing cancer lines) after which MTT assay was performed. Spectrophotometric readings at 590 nm and 630 nm using a 96-well plate reader were used to establish growth and viability of cells. Each drug dose was tested in quadruplicates and experiments repeated twice.

IMR90, WI38, normal human neonatal keratinocytes (PCS-200–010), H460, A549, U2OS, mouse embryonic fibroblasts (MEFs, SCRC-1040), telomerase-immortalized human bronchial epithelial HBEC3 (CRL-4051), Daudi, HCT116, Ramos, Raji and CA46 were obtained from ATCC. RCC4 cells were gift from Dr. W. Kaelin. H460 and lymphoma lines Daudi, Ramos, Raji and CA46 were maintained in RPMI-1640 medium (Gibco, 11875–119). IMR90, WI38, MEFs, RCC4 and U2OS cells were maintained in DMEM medium (Gibco, 12430–062). A549 and HCT116 cells were grown in F12-K (ATCC, 30–2004) and DMEM/F12 medium (Gibco, 11320–033), respectively. All media were supplemented with 10% fetal bovine serum (Rocky Mountain Biologicals, FBS-BBT-5XM) and 1% Pen-Strep solution (100 Units/ml penicillin and 100 μg/ml streptomycin) (Gibco, 15140–122). Keratinocytes were grown in Dermal Cell Basal Medium (ATCC, PCS-200–300) supplemented with Keratinocyte Growth Kit (ATCC, PCS-200–040). HBEC3 cells were propagated in Airway Epithelial Cell Basal Medium (ATCC, PCS-300–030) supplemented with Bronchial Epithelial Growth Kit (ATCC, PCS-300–040). All cells were grown at 37°C in a 5% CO₂ humidified atmosphere containing either 5%O₂ (IMR90, WI38 and MEFs) or 20% O₂ (all other cells).

Cells were grown in Airway Epithelial Basal Media supplemented with Bronchial Epithelial growth kit from ATCC. Cells were transfected with Lipofectamine or HIPERFECT for siRNA experiments and cell lysis performed 24 and 48 hrs after transfection. For imaging studies, cells were grown to 50 or 100% confluency in Lab-TekII 8-chamber slides (Fisher), transfected, and growth for further 24–48 hrs in media.