Figure 7: Mitosphere formation is not linked to Parkin-related mitophagy.

(a,b) OS led to an upregulation of LysoTracker Red (LT)-positive structures as investigated via flow cytometry (a) and fluorescence microscopy (b) in aSyn and Ctr H4 cells treated with 300 μM H₂O₂ for 4 h. (a) The relative mean cellular intensity of the LT signal compared to VC-treated Ctr H4 cells is shown (Two-way ANOVA Bonferroni’s Multiple Comparison Test, n = 3, F = 131.5, p < 0.0001 (VC vs. H₂O₂); F = 29.68, p = 0.0006 (Ctr vs. aSyn)). (c) MTdr-stained mitospheres did not show co-localization with LT-positive organelles as investigated via fluorescence microscopy 4 h after the treatment with 300 μM H₂O₂. Intensity profiles, along the white arrows, of the MTdr (red) and LT (green) staining are depicted. (d–f) H4 cells transiently overexpressing Parkin were treated for 4 h with 300 μM H₂O₂ or 50 μM CCCP as control. Parkin (d) and p62 recruitment (e) as well as co-localization of mitochondria and LT-positive organelles (f) was investigated via fluorescence staining. In contrast to mitochondria of CCCP-treated cells, H₂O₂-induced mitospheres did not show co-labeling with Parkin, p62 or LT. The red color of the intensity profiles along the white arrows reflects the MT or MTdr staining, the green color represents Parkin, p62 and LT staining in (d–f) respectively.