Cytotoxic synergism of pemetrexed followed by ITF2357 in human NSCLC cell lines. (A) Analysis of cell viability by MTT assay in the indicated NSCLC cell lines treated for 72 h with increasing concentrations of ITF2357. (B) Western blot analysis of acetylated histone H3 (Ac-H3), acetylated α-Tubulin (Ac-Tubulin) and TS protein expression in total cell lysates from the indicated NSCLC cell lines treated with increasing concentration of ITF2357 for 24 h. HSP72/73 expression was used as loading and transferring control. Western blots representative of two independent experiments with similar results are shown. (C) TS mRNA expression by quantitative RT-PCR in NSCLC cells treated with increasing concentration of ITF2357 for 24 h. Results are presented as the mean ± SD of two independent experiments. p values were calculated between control and treated cells (*p < 0.05). (D) Analysis of cell viability by MTT assay in the indicated NSCLC cell lines treated with ITF2357 and pemetrexed (Pem, drug ratio 1:1) alone or in combination. Treatment with pemetrexed for 24 h was followed by treatment with ITF2357 for 48 h. (●, ITF2357; ■, pemetrexed; ▲, combination). (A,D) The results are reported as "viability of drug-treated cells/viability of control cells" × 100 and represent the mean ± SD of three independent experiments performed in triplicate. (E) Interaction between pemetrexed followed by ITF2357 treatment evaluated on the basis of the Combination Index, which is plotted against fractional growth inhibition. Data are means of triplicates from experiments that were repeated three times.