Effect of etomoxir and ATO on cell viability, cycle phase distribution and apoptosis generation in HL60 cells.

<p>Cells were incubated for 24 h with the indicated concentrations of etomoxir (Eto) and ATO, alone and in combination. When nothing is indicated, ATO was used at 2 µM. For convenience, the drug concentrations are sometimes indicated as subheadings. When indicated, the cells were co-incubated with the pan-caspase inhibitor z-VAD-fmk (50 µM). (A) Changes in cell viability, as evidenced by the MTT assay. Absorption values are indicated in relation to untreated (Cont) cultures. (B) Frequency of cells at the different phases of the growth cycle, namely G₁, S and G₂/M, and with sub-G₁ DNA content (apoptotic). Examples of flow cytometry histograms are presented in (E). (C) Frequency of apoptotic cells, as determined by flow cytometry. (D) Caspase-3 cleavage/activation, determined by immunoblot. β-actin is included as a loading control. The bar charts in (A–C) represent the mean ± S.D. of at least three determinations. Symbols mean: (*) significant differences between the indicated pair values; (^#) significant differences between the combined treatment and the sum of values in the corresponding individual treatments (e.g., co-incubation with 100 µM Eto and 2 µM ATO, in relation to the sum of 100 µM Eto alone plus 2 µM ATO alone) (n.s., non-significant). To better discern differences, in this case the sum of values in individual treatments is indicated by a horizontal white line within the bar corresponding to the combined treatment. Single symbol, <i>p</i><0.05; double symbol, <i>p</i><0.01; triple symbol, <i>p</i><0.001.