Fig. 4: Influence on mitochondria, lysosomes/autophagy, ER and extracellular flux analysis.

A, B Mitochondrial function and/or acidic compartments (LysoTracker and Acridine orange (AO)) in 2D-cultured and treated GBM cells (HROG05, HROG63) was examined by immunofluorescence imaging as described in materials and methods (Mito- [red] and Lyso-Tracker [green]; Calcein AM [green] and Acridine orange [orange]). A Merged fluorescence is presented (scale bar A: 50 μm). CDKi-SpyADI impairs mitochondrial membrane potential (MMP) and SEQ-abemaciclib administration elevates LysoTracker and AO staining. B Quantification of Mito- and Lyso-Tracker as well as AO-positive cells using ImageJ software (integrated density analysis). n = 3–6 independent experiments; **p < 0.01; ***p < 0.001; §§§p < 0.001; $p < 0.05; $$p < 0.01; $$$p < 0.001. One-way ANOVA. C–H The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of treated HROG05 cells was recorded by a Seahorse XFe24 Analyzer (C, D). To determine the basal respiration (basal, E), ATP-dependent respiration (ATP, F), proton leak (G) and coupling efficiency (H) mitochondrial stress test was applied with injection of 1.5 µM oligomycin (Port A), 1.0 µM FCCP (Port B) and 0.5 µM Antimycin A and Rotenon, each (Port C). Decreased OCR and ECAR after treatment with abemaciclib, SpyADI or SEQ dinaciclib/SpyADI were observed. Significant reduction of basal OCR and partially also of ATP-production-based OCR was shown, indicating dysfunctional mitochondria. n = 3 independent experiments; ∗p < 0.05; ∗∗p < 0.002; ∗∗∗p < 0.0002; ∗∗∗∗p < 0.0001. One-way ANOVA.