Cells were derived from a single tumor, and subsequently

induced to differentiate into the epithelioid (STAV-AB) and the sarcomatoid phenotype (STAV-FCS), respectively, by altering the serum composition [30]. Hence, STAV-AB cells were grown in Gibco RPMI 1640 medium (Invitrogen) and Alvocidib concentration 10% human AB serum, whereas STAV-FCS cells were grown in the same medium and 10% fetal calf serum. The specific differentiation of these cells has been evidenced by immunoprofiling showing that STAV-AB cells express more cytokeratin, whereas STAV-FCS cells have stronger reactivity to vimentin antibodies [21] as well as by morphometry. The elongated sarcomatoid cell INCB018424 supplier morphology of the STAV-FCS cells and the more round epithelioid morphology of the STAV-AB cells have been confirmed by average length:width ratios of 3.42 in the STAV-FCS cells and 1.58 in the STAV-AB cells [31]. Jurkat cells were obtained from the American

Type Culture Collection (ATCC) and grown in RPMI 1640 medium and 20% fetal calf serum. All cells were grown at 37°C with S3I-201 solubility dmso 5% CO2 and passaged approximately twice per week. Treatment of cell cultures and inhibition of signalling enzymes To investigate the contributions of several signalling pathways, inhibitors were used against key enzymes. Cells were washed, trypsinized and re-seeded with the respective inhibitors (specified in table 1) 24 h prior to selenite treatment, except for the JNK-inhibitor, with which they were pre-incubated for 1 h. Selenite was then added to the medium and the cells Celastrol were harvested 24 or 48 h later. Titrations were performed with all inhibitors based on the manufacturers’ instructions and concentrations reported in the literature. In all cases, the highest non-toxic doses tested were accepted. Table 1 Chemical inhibitors against apoptosis signalling enzymes Inhibitor Target Concentration Purchased

from SB 203580 p38 5 μM Merck SP 600125 JNK 10 μM A.G. Scientific Pifithrin p53 10 μM A.G. Scientific Pepstatin A Cathepsin D, E 5 μM Calbiochem Ca-074 Me Cathepsin B 10 μM SERVA Electrophoresis GmbH Cell viability assays Viability assays were performed in conjunction with flow cytometry experiments to obtain internal controls. Aliquots of cell suspensions prepared for flow cytometry were plated in triplicates in 96-well plates, with a density of approx. 5000 cells per well. They were then analysed using the WST-1 assay (Roche), whereby a tetrazolium salt is cleaved by mitochondrial enzymes to yield a coloured product, to measure viability. The plates were read in a Spectramax spectrophotometer at 450 nm with subtraction of background absorbance at 600 nm. Flow cytometric analyses Flow cytometric assays for detection of apoptosis were carried out using the Annexin V kit (Caltag Laboratories) according to the manufacturer’s protocol.