Figure 3: H₂O₂-induced mitospheres form in a Drp1-dependent manner and are linked to changes in the levels of the fusion proteins MFN1 and OPA1.

(a) H4 cells were treated with H₂O₂ or VC for 4 h with (Drp1 inhibition) or without Mdivi pretreatment (control) and mitochondria were stained via MT. (b) The quantification of the percentage of cells with mitospheres shows that Drp1 inhibition substantially reduced mitosphere formation (Two-way ANOVA Bonferroni’s Multiple Comparison Test, n = 4 with >160 cells quantified per sample, F = 51.39, p < 0.0001 (control vs. Drp1 inhibition)). (c) H4 cells were transiently transfected with vectors expressing shDrp1 or a scrambled shRNA as control (shScr) and Drp1 levels were detected via WB. Considering a transfection efficiency of ~20–30%, the reduction in Drp1 protein levels in shDrp1 transfected cells by ~20% supports a strong downregulation of Drp1 in shRNA expressing cells. Fold protein levels per mock-transfected cells are shown (unpaired two-tailed t-test, n = 3, p = 0.0089). (d) H4 cells transfected with shDrp1-GFP constructs show more elongated mitochondria than control-transfected cells (shScr-GFP). Mitochondria were stained with MT. (e) Mitosphere formation was reduced in shDrp1-GFP expressing H4 cells treated with 300 μM H₂O₂ for 4 h as compared to shScr-GFP expressing control cells. (unpaired two-tailed t-test, n = 3 with >120 cells quantified per sample, p = 0.0086) (f) Protein levels of Drp1 and Fis1 were investigated via WB in H4 cells treated with VC or H₂O₂ (300 μM, 4 h; unpaired two-tailed t-tests, Drp1: n = 3, p = 0.3026; Fis1: n = 6, p = 0.1577). (g) Three MFN1 bands were observed in the mitochondrial fraction (Hsp60 ↑, TOM20 ↑, β3tubulin ↓) of H4 cells in a molecular weight range from 60 to 90 kDa. OS led to a strong reduction in the level of the MFN1 band detected at ~60 kDa (3) as analyzed in H4 cells exposed to 300 μM H₂O₂ for 4 h (unpaired two-tailed t-tests, n = 7, p = 0.8011 (1), p = 0.0866 (2), p = 0.004 (3)). H4 cells treated with 300 μM H₂O₂ for 4 h show a significant reduction in the levels of the long OPA1 isoforms (1 and 2) and an increase in the two short isoforms (4 and 5; unpaired two-tailed t-tests, OPA1: n = 3, p = 0.1749 (total), p = 0.0467 (1), p = 0.0001 (2), p = 0.5515 (3), p = 0.0081 (4), p = 0.0417 (5)).