Figure 4
Calcium transport inhibition assays with KB-R7943. A – C. Ca2+ - dependent luminescence at increasing concentrations of KB-R7943. Cells were incubated for 30 minutes in 2% (v/v) DMSO (concentration of solvent in the culture with the highest concentration of KB-R7943) or in increasing concentrations of KB-R7943. The profiles for the first 30 seconds are presented for 12.5 μM, 25 μM, 50 μM, 100 μM and 200 μM of KB-R7943. CaCl2 (1 M, 0.1 ml) was added at 5 seconds to wells containing 0.1 ml of cells (0.1 OD600) and the signals recorded for 60 seconds. RLUs were normalized against the RLU of the 5 second point for that group ( RLUt (0 .. 30)/RLUt = 5 ). Data represent the averages of four experiments. D – E. Inhibitory concentrations for KB-R7943. Inhibitory assays were performed with nine concentrations at two-fold dilutions from 0.2 mM KB-R7943. Y axis: the areas under the curve (AUC) from 4 to 16 seconds for the DMSO samples were divided by the AUC for the different concentrations of KB-R7943 in each experimental group and multiplied by 100 to express the fractions as percentages. The data were fitted and the IC50 and slopes inferred with the following equation: f(x) = A2 + (A1 – A2)/(1 + (x/L) S ). Where “A2” is the limit to the left of the sigmoid (highest signal, lowest concentration), “A1” is the limit to the right (lowest signal, highest concentration), “L” is the concentration equivalent to half of the signal of the left limit (IC50), and “S” is the slope. Data represents the averages and standard deviations of four different experiments.