Variance was stabilized using base-2 logarithmic transformation.
Basic data preprocessing was done in Partek Genomics Suite 6.6 (6.12.0712) with these options: Background correction - RMA algorithm, Normalization - Quantile normalization, Log Probes using Base - 2, Probeset summarization - Median polish.
Fragmented cRNA was hybridized in GeneChip 640 Hybridization Oven (Affymetrix) to a Human Gene 1.0 ST array (Affymetrix) according to standard protocol. The washing and staining of the arrays with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR) was completed in Fluidics Station 450 (Affymetrix). Arrays were scanned using GeneChip 3000 7G Scanner (Affymetrix).
Total RNA was amplified and labelled using standard protocols for Affymetrix GeneChip® Human Gene 1.0 ST Array.
Multiple myeloma cells (MM) MOLP8, (DSMZ, Braunchweig, Germany) were cultivated in RPMI-1640 medium supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 20% fetal bovine serum (FBS; PAN, Germany). Cells were maintained at 37°C in a humidified atmosphere containing addition of 5% CO2 and consisting of 95% air.
MOLP8 cells were treated for 24 h by HDAC inhibitor SAHA ([syn. Vorinostat; Zolinza] Cayman Chemicals, USA, #10009929). We used final concentration 70 nM of SAHA that was dissolved in 96% ethanol. Concentration of SAHA was selected according to cell number which was reduced to 50% after treatment (Foltankova et al., 2012). The cells were treated 24 h after cell seeding in density 2e-5/ml. After 24 h of SAHA treatment the cells were harvested for RNA isolation and subsequent cDNA microarrays.
Total RNA was isolated using RNAqueous-4PCR kit (Ambion, Applied Biosystems, #AM1914) according to the manufacturers' protocols. Quality and concentration of RNA was measured by NanoDrop 2000 spectophometer (Thermo Scientific, USA). RNA integrity was assessed on Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). All RNA samples had RIN above 8.
Human leukemia K562 (European Collection of Cell Cultures, ECACC, UK) were cultivated in RPMI-1640 medium supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 20% fetal bovine serum (FBS; PAN, Germany). Cells were maintained at 37°C in a humidified atmosphere containing addition of 5% CO2 and consisting of 95% air.
Leukemia K562 cells growing 24 h after seeding were treated by HAT inhibitor Anacardic acid Derivative (MG149 088) (Ghizzoni et al., 2012) and we found optimal concentration of MG149 according to the test of cell viability (trypan blue or eosin staining) and according to growth curves. Optimal concentration that reduced cell viability to 70% and suppressed proliferation to ~50% was 20 microM. As a solvent for MG149 we used DMSO. Control cells were affected by relevant concentration of DMSO, but no changes in the cell proliferation were observed in comparison with non-treated cells (Not shown). Twenty four hours after the cell treatment by 20 microM MG149, the cells were harvested for analysis by cDNA microarrays.