Fig. 3

Cytotoxic effects of MitA and SpyADI combination. A Bliss independence calculation after combination treatment of GBM cell lines HROG02, HROG05, HROG52, and HROG63. Synergistic effects were obtained in 3/4 cell lines by applying the SIM combination approach. B γ-H2AX staining [red] of HROG05 and HROG63 treated with SpyADI and SIM combination with [ +] and without [−] radiation. Scale bar as indicated: 50 µm. Nuclei were counterstained with DAPI. Images were taken on a Zeiss Elyra 7 Confocal Laser Microscope. Representative images of n = 3 independent experiments. C HROG cells received 10 × 72 h consecutive mono- and SIM combination therapy cycles and stained with crystal violet to identify long-term treatment effects. Viability reduction (%) after treatment (HROG05: dark purple; HROG63: light purple) was quantified by normalization to control values (untreated cells, set to be 100%). n = 3 independent experiments. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. control; #### p < 0.0001 vs. monotherapy; $$$$ p < 0.0001 vs. SEQ-combination, Two-way ANOVA (Tukey's multiple comparisons test). D Viability in the 3D model in mono- and SIM combination regimens (HROG05 [black], HROG63 [grey]) was assessed with CellTiter-Glo 3D. n = 3 independent experiments. **p < 0.01; ****p < 0.0001 vs. control; ##p < 0.01 vs. monotherapy, Two-way ANOVA (Tukey's multiple comparisons test)