Cell lines
The human BL cell line Akata was cultured in RPMI 1640 medium (Invitrogen, Basel, Switzerland), supplemented with 10% heat-inactivated fetal calf serum, L-Glutamine (1%), penicillin (1 U/ml), streptomycin (1 μg/ml). The human bladder carcinoma cell line T24 was cultured in McCoy's medium supplemented with 10% fetal calf serum, L-Glutamine (1%), penicillin (1 U/ml), streptomycin (1 μg/ml). The cells were grown at 37°C in 5% CO2 humidified atmosphere and split every third day. Akata cell line was a kind gift from Dr. A. Bell (Birmingham, UK), and T24 cell line was a kind gift from Dr. H. Wunderli-Allenspach (ETH Zürich, Switzerland). The optimal cell density (5 × 105 cells/ml) for experiments with Akata cells allowed logarithmic cell growth over the observation period.
DNA-methylase inhibitor treatments
5-Azacytidine was purchased from Sigma-Aldrich Chemie Gmbh (Buchs, Switzerland) as lyophilized powder and stored at -20°C. 5-Azacytidine solution was prepared fresh for each experiment in PBS at a concentration of 2.4 mg/ml (10 mM) and sterile filtered. Zebularine was purchased from Calbiochem (Merk Biosciences, Darmstadt, Germany) and stored at 4°C. Zebularine was dissolved in PBS at a concentration 28.5 mg/ml (120 mM), sterile filtered and stored at 4°C. For each experiment with Akata cells, viable cells were counted by trypan-blue exclusion method and resuspended in fresh medium at 0.5 × 106 cells/ml. T24 cell line experiments were performed as described [18].
Quantitative real-time PCR analysis
RNA isolation was then performed by using Rneasy Mini Kit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturer's protocol. Contaminating genomic DNA was removed by using DNA-free (Ambion Europe, Huntingdon, UK) according to the manufacturer's protocol. The RNA concentration was measured with an Eppendorf Bio Photometer (Vaudaux Eppendorf, Dübendorf, Switzerland). cDNA was prepared by reverse transcription of total RNA using the Omniscript RT-Kit (Quiagen) following the manufacturer's protocol. qRT-PCR was performed in a reaction volume of 10 μl with the ABI-TaqMan Master Mix with uracil-N-glycosylase (Applied Biosystems, Rotkreuz, Switzerland). Sequence information for the Taqman systems will be furnished upon request. mRNA expression of the target genes was normalized to the expression of the housekeeping gene HMBS [30]. The normalized transcription values correspond to 2-CT(EBV)-CT(HMBS) = 2-ΔCT, where CT is the cycle threshold number that quantifies the target present.
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