77 mM 0.77 mM 0.77 mM SAHA 0.16 μM 0.16 μM 0.16 μM Abacavir 0.11 mM 0.11 mM 0.11 mM Retinoic acid 0.25 μM 0.25 μM 0.25 μM Resveratrol 15 μM 15 μM 40 μM Clonogenic survival For clonogenic assays, cells were treated with/without 3 μM (D283-Med) or 5 μM (DAOY, MEB-Med8a) 5-aza-dC in cell PLK inhibitor culture flasks for three days. Subsequently, medium was renewed and supplemented with 5-aza-dC and 15

μM (D283-Med, DAOY) or 40 μM Resveratrol (MEB-Med8). After three days, cells were counted, seeded at three different cell densities in duplicates in 6-well cell culture plates, and normal medium without mediators was added. Ten to 14 days later, colonies were washed with PBS, fixed with ice-cold ethanol/acetone (1 : 1) for 10 min, CHIR98014 order stained with Giemsa solution (1 : 1 with distilled water) for 5 min, and washed with distilled water. Colonies with > 50 cells were counted indicating plating efficiency (PE). The ratio between PE of treated cells and PE of untreated cells represented the surviving fraction (SF) of clonogenic cells. Statistics Statistic analyses of were performed using the parametric, one-way, and paired Student’s t-test with Microsoft Excel 2003 software. P-values ≤ 0.05 (*) were considered as statistically significant and p-values ≤ 0.001 (**) as highly statistically significant. Detailed drug interaction analyses regarding

synergistic or additive effects were conducted using the Bliss independence (BI) model Lenvatinib ic50 which is based on the non-interaction theory. The BI model compares the estimates of the combined effects calculated on the individual drug effects with those obtained from the experiment. Therefore, the following equation was used: E i = E A × E B , where E i is the estimated amount of metabolic activity of the theoretical combination of substance A and B, and E A and E B are the experimental rates of metabolic activity of each drug alone. The interaction of both is described by the difference ΔE between the estimated and the observed rates of metabolic activity ΔE = E estimated − E observed [35]. The non-parametric

approach described by Prichard et al. was modified and used to calculate statistical significance of synergism. Fenbendazole In each of the three independent experiments, the observed rates of metabolic activity were subtracted from the predicted values, and the average difference of each experiment was calculated. Statistically significant synergy was claimed when the average difference as well as its 95% confidence interval was positive [36]. Results and discussion To determine submaximal concentrations for the inhibition of the metabolic activity of MB cells, we performed incubation experiments with the single drugs. The mean drug concentration of the three examined cell lines which inhibits the metabolic activity by 30% (IC30) was chosen for combination treatments with 5-aza-dC for three days (Table 1).