Mitochondrial but not cytosolic ROS contribute to thrombopoiesis. MKs were incubated for 5 hours with the indicated drugs (apocynin 1 mM; VAS2870 15 µM; MitoTEMPO 10 µM) or dimethyl sulfoxide solvent (0.1%) as the control. (A,C) The percentage of proplatelet-forming MKs relative to total cells observed by phase-contrast microscopy. Dots show individual data points from 3 independent experiments. Means ± SEM; n = 3; ratio paired Student t test. **P ≤ .01. (B) Cells were incubated with DHE fluorescent dye before being processed by flow cytometry; relative mean fluorescence intensity expressed as fold change over control cells. Means ± SEM; n = 4; ratio-paired Student t test. *P ≤.05. (D) Representative micrographs of MitoTEMPO-treated or untreated cells obtained with phase-contrast or MitoSOX fluorescence microscopy, as indicated (stars, round MKs; arrowheads, intermediate MKs; arrows, proplatelet-forming MKs). Bar represents 10 µm. (E-F) Transduced CD34⁺-derived mature MKs with the indicated lentiviral particles were treated with doxycycline (0.3 µg/mL) for 7 hours to induce transgene expression. Where indicated, MitoTEMPO (10 µM) was added. MKs were then directly analyzed without the BSA gradient. (E) GFP⁺ proplatelet-forming MKs were counted relative to total GFP⁺ cells (means ± SEM; ratio paired Student t test; EV vs DN; n = 6; MitoTEMPO vs no MitoTEMPO; n = 3). *P < .05; ^##P ≤ .01. (F) Cells were incubated with MitoSOX fluorescent dye. Mean fluorescence intensity was measured by flow cytometry in GFP⁺/CD42⁺ cells. Data are expressed as fold change of induced relative to noninduced condition; ratio paired Student t test. *P < .05; n = 3. EV, empty vector.