Effect of ST1926, BAPTA, and the combination of the 2 agents on the levels of intracellular calcium, apoptosis, and caspase-3 activation in NB4 cells. (A) Following preloading with the calcium fluorescent indicator FURA-2, NB4 cells (1 × 106/mL) were resuspended in calcium-containing PBS. Cells were placed in a cuvette under stirring at 37°C and changes in fluorescence were measured continuously with the use of a spectrophotofluorometer. NB4 cells were incubated with the indicated concentrations of BAPTA prior to addition of ST1926 at the concentration of 1 μM. The left arrow indicates the addition of BAPTA, whereas the right arrow indicates the addition of ST1926. Each tracing is representative of at least 2 independent experiments run in triplicate. (B) NB4 cells (150 000/well) were treated with vehicle, the indicated concentrations of BAPTA, ST1926 (1 μM), or CD437 (1 μM), and mixtures of the intracellular calcium chelator and RRMs for 6 hours. Following treatment, aliquots of the cultures were subjected to the determination of the apoptotic index by scoring the percentage of cells showing morphologic signs of nuclear fragmentation upon DAPI staining. The results are expressed as the mean ± SD of 3 replicate cultures. The data are representative of 2 independent experiments. *Significantly lower than the corresponding ST1926- or CD437-treated group (P < .01 according to the Student t test). (C) Cells were treated for 6 hours with BAPTA (50 μM or 100 μM) in the presence of medium or medium containing 1 μM ST1926. The level of caspase-3 was measured following incubation of cell extracts with the fluorogenic peptide substrate DEVD-amc. The results are expressed as the mean ± SD of 3 replicate cultures. *Significantly lower than the corresponding ST1926- or CD437-treated group (P < .01 according to the Student t test).
