Setup of the curve shift synergy experiments.

<p><b>A</b>: First, concentrated mixtures are made of the single agents. Subsequently, dose response curves are generated. For the overlay, all concentrations are scaled relative to the measured IC₅₀s of the single agents. The example shown is the effect of the PI3-kinase inhibitor GDC-0941 (green) and the MEK inhibitor AZD-6244 (blue) on proliferation of the HCT 116 cell line. Mixture ratios used were 1:1, red; 4:1, orange; 1:4, yellow. <b>B</b>: Calculation of the Combination Index (CI). The fraction affected <i>(F)</i> is equivalent to 1/100 of the %-effect. If CI < 1, compounds show synergy. The fitted CIs at <i>F</i> = 0.5 (50% effect), for all mixtures, are reported as CI_0.5. <b>C</b>: Calculation of the isobologram [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125021#pone.0125021.ref007" target="_blank">7</a>]. Single agent concentrations needed to achieve 75% effect in the cell proliferation assay are displayed in blue dots and connected by the blue line. The concentrations where the combination curves achieve 75% growth effect are displayed in red, yellow and orange, where the x and y coordinates are the respective component concentrations. If the combination points lie below the blue line, there is synergy. <b>D</b>: Reproducibility of CI_0.5 measurements in a positive control of AZD-6244 / GDC-0941 (light bars, average 0.33, SD: 0.06, n = 12) and a negative control of doxorubicin / doxorubicin (dark bars, average 1.04, SD: 0.16, n = 15). Both were combined in the HCT 116 cell proliferation assay. Every bar represents an individual mixture ratio (yellow, red, orange) that was tested in duplicate. <b>E:</b> Curve shift experiment of the AZD-6244 / GDC-0941 combination in the RPE-1 fibroblast cell line, which is immortalized using hTERT. Here, the combination also shows synergy.