Fig. 3. TRAILR2 expression is essential for TRAIL hypersensitization within tumor cell spheroids.

a, b TRAILR surface expression. HCT116/NCI-H460 wildtype (WT) and HCT116/NCI-H460 TRAILR2 knockout (T2 k/o) cells were analyzed by flow cytometry. Histograms are representative of two independent experiments. c, d, f, g HCT116/NCI-H460 cells or HCT116/NCI-H460 TRAILR2 knockout (T2 k/o), grown in 2D or as spheroids (day 11) were stimulated with TRAIL for 6 h. In (d, g), cells were detached, or spheroids were dissociated prior to treatment. Loss of viability was measured flow cytometrically by Annexin V-EGFP staining. Data shown are mean values ± SEM from three independent experiments, with exception of HCT116 T2 k/o cells in (d), where bars indicate range (n = 2). The experiment shown in (f) was performed side by side with the experiment shown in supplementary Fig. 5c, thus the curves derived from WT cells (2D and 3D) are identical. The same is true for the data shown in (g) and in supplementary Fig. 5d. Insets serve as illustrations. e, h EC₅₀ values obtained from nonlinear regressions (sigmoidal dose response). Minus sign indicates cases in which the EC₅₀ could not be calculated.