Coupling of mitochondrial dynamics and mtROS levels during formation of proplatelets. Mature MKs were treated for 5 hours with the indicated drug (Mdivi-1, 10 µM; NAC, 1 mg/mL; MitoTEMPO, 10 µM) or dimethyl sulfoxide solvent as the control, and processed for fluorescence microscopy (A), phase-contrast microscopy (B-C), and fluorescence confocal microscopy (D). (A) Mean MitoSOX fluorescence intensity (left) of individual round (red dot), intermediate (blue dot), and terminal (green dot) MKs upon treatment with Mdivi-1. Dot plots show individual data and median lines (n = 3, control vs Mdivi-1; Mann-Whitney U test; ***P < .001). Representative images (right), as indicated (arrowheads: intermediate MKs). Bar represents 10 µm. (B) Distribution of the indicated MK subtypes, expressed relative to total cells observed. (Means ± SEM; n = 3; ratio paired Student t test for drug-treated vs solvent-treated MKs). *P < .05. (C) Transduced CD34⁺-derived, mature MKs with the indicated lentiviral particles were treated with doxycycline for 7 hours to induce transgene expression and with Mitotracker during the last 30 minutes. MKs were then directly analyzed without the BSA gradient. GFP⁺, proplatelet-forming MKs were counted and expressed relative to total GFP⁺ cells (means ± SEM; n = 3; ratio paired Student t test DRP1^SD vs EV); *P < .05. (D) DMSO solvent- (control) and MitoTEMPO-treated MKs were analyzed by fluorescence confocal microscopy for colocalization of MitoTracker and pSer⁶¹⁶-Drp1. Pearson’s correlation coefficients are shown for individual MKs from each subtype. Dot plots show individual data and median lines (n = 3; Mann-Whitney U test). **P < .05; ***P < .001. NS, not significant.