"Another Basel Cell Carsinoma.
Both cell lines were grown in Dulbecco's modified Eagle medium (DMEM) (4.5 g/ l glucose) and Ham F12 (F12) medium (1 1) supplemented with 2 mM l-glutamine and 10%% fetal calf serum (FCS) (GIBCO, Basel, Switzerland) (cell culture medium) in a humidified atmosphere of 95%% air and 5%% CO2 at 37 °C.
In some experiments, BM specimens of 6 CML patients at diagnosis were cultured in vitro for 3 to 14 days with 5 µM of the purified imatinib mesylate (Novartis Pharma AG, Basel, Switzerland, MTA31812); cells were recovered at day 3, 5, 7 and 14 for RNA extraction.
Transfections were performed using Mammalian Epithelial Cells Amaxa Basic Nucleofector Kit (Lonza, Basel, Switzerland) and cells were electroporated according to manufacturer's instructions.
The pellet was resuspended in 1 ml of 0.1 M sodium phosphate buffer (pH 8.0), 1.25 ml of glass beads were added (diameter of 0.5 mm, Willy A. Bachofen AG. Basel, Switzerland), and the cells were mechanically disrupted by vortexing for 20 min. The debris of cells was removed by centrifugation and the activity of PGA was measured in the supernatant.
24 Fresh human aortic endothelial cells (Lonza, Basel, Switzerland) were cultured in EGM2 medium supplemented with 3% fetal bovine serum (Lonza, Basel, Switzerland), and seeded onto six-well tissue culture plates after two passages.
Primary human small airway epithelial cells (SAEC, Lonza Group, Basel, Switzerland) were grown as monolayer in 5% CO2 at 37°C in SAGM (Lonza Group, Basel, Switzerland) with 50 μg/ml penicillin, 50 mg/ml streptomycin (Sigma, St . Louis MO) and 10% heat-inactivated FBS (Sigma, St . Louis MO).
MRMT-1 rat mammary gland carcinoma cells (Novartis Oncology Research, Basel) and H1299 human lung cancer cells were cultured in RPMI 1640 (Gibco) medium, human SY5Y cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM).
Additionally, PREC cells (Lonza, Basel, Switzerland) were used as a normal human prostate cell line control.
For nucleofection (AmaxaNucleofector, Lonza, Basel, Switzerland), 5 million cells were used per reaction.
