2.4. Caspase 3/7 Activation Assay

To gauge the effects of SMI on cell death by apoptosis, caspase 3/7 activity as surrogate marker was measured in a cell-based assay based on the luminogenic substrate Z-DEVD-aminoluciferin with a tetrapeptide sequence specific for caspase-3/7. Therefore, cells were seeded in 96-well plates (3 × 10³ cells/well) in complete culture medium. On the next day, either an inhibitor or the solvent was added at the indicated doses (Figure 2), and the incubation was continued for an additional period of 6 h. Afterwards, caspase activity was measured following the instructions of the manufacturer (Promega). Briefly, the cell culture plates were removed from the incubator, and the luminogenic substrate was added. Assay plates were incubated at 22 °C for 1 h before recording the luminescence with a GloMax microplate reader. For the generation of caspase 3/7-positive control cultures, cells were treated with 1 μM staurosporine (Sigma-Aldrich, Taufkirchen, Germany) for 2 h, and caspase activity was determined as described above (Supplementary Figure S2).

Effects of SMI on caspase 3/7 activity of glioblastoma cell lines. Glioblastoma cells were challenged with (A) dactolisib, (B) ipatasertib, (C) MK-2206, (D) regorafenib, (E) trametinib, or solvent control for 6 h. Afterwards, the cells were subjected to caspase 3/7 enzyme activity quantification. Data are presented as mean ± SEM (n ≥ 8 separate cultures for caspase activity assay); * p < 0.05 versus control cultures (Kruskal–Wallis test with post hoc Dunn’s test).