Fig. 1: Viability and cytotoxicity assays after mono- and combination therapy using different settings and experimental conditions.

A Viability of non-malignant control cells. Cells (L929, NHDF, and h-MSCs) were treated with the respective substances for 2 × 72 h in a sequential treatment regimen (SEQ). Quantitative analysis was done using Calcein AM assay. n = 3 independent experiments. B–E GBM cells were treated with the respective substances for 2 × 72 h in a simultaneous or sequential treatment regimen (SIM; SEQ). B Quantitative analysis of cell viability using Calcein AM in the 2D-model (HROG02, HROG05, HROG63). n = 8 independent experiments. C Bliss independence calculation. The Bliss independence model was applied to determine synergistic, additive or antagonistic effects based on the results obtained after 2 x 72 h treatment with the indicated substances. Interpretation is as follows: Δ < 1: synergism (green pattern); Δ = 1: additive (yellow pattern); Δ > 1: antagonistic (orange and red pattern). Image was created with Biorender.com. D Quantitative analysis of cytotoxicity was done via flow cytometry upon staining with Yo-Pro1 and PI. Cells were either characterized as early-apoptotic, late-apoptotic, or necrotic. n = 3 independent experiments; statistical differences were obtained for the necrotic cell fraction: *p < 0.05; ***p < 0.001; §p < 0.05; $$p < 0.01; #p < 0.05. E Quantitative analysis of cell viability in the 3D systems (HROG05, HROG63) was done using 3D-Glo. B, E Viability reduction (%) after treatment was quantified by normalization to control values (untreated cells set to be 100 %). n = 8 independent experiments; §§p < 0.01; §§§§p < 0.0001; $p < 0.05; $$$p < 0.001; $$$$p < 0.0001; ##p < 0.01; ####p < 0.0001. One-way ANOVA.