OxPhos inhibition induces ROS and mitophagy in OCI-AML3 cells cocultured with MSCs. (A) Summary data (left) and representative flow cytometry histograms (right) showing ROS production in OCI-AML3 cells treated with the OxPhosi IACS-010759 (30 nM) for 72 hours with or without cocultured MSCs. Comparisons of the increase in ROS by treatment with IACS-010759 (IACS) between the single-culture condition and MSC coculture condition are shown in the middle. An increase in relative fluorescence is reflected by a rightward shift along the x-axis of the histograms. (B) Cells were treated with IACS-010759 (30 nM) for 18 hours in the absence or presence of the lysosomal inhibitor bafilomycin A1 (10 µM), and cell lysates were examined by immunoblotting. LC3-II protein levels were normalized to LC-I levels to determine the differences in protein expression between the cells cultured with and without MSCs. (C) Uptake of mitochondria by lysosomes was evaluated by mitophagy assay. Comparisons of the increase in mitophagy dye positivity by treatment with IACS-010759 between the single-culture condition and MSC coculture condition are shown on the right. (D) Representative immunoelectron microscopy images showing MSC-derived mitochondrial fragments inside autophagosomes in cocultured OCI-AML3 cells. OCI-AML3 cells were cocultured with PDHA1-dsRed–transfected MSCs and treated with 30 nM IACS-010759. Electron microscopy images at ×2000 magnification. Fixed sections were immunolabeled with gold as described in supplemental Materials and Methods. Black arrows indicate mitophagy; red arrows indicate MSC-derived mitochondrial fragments surrounded by autophagosomes. Error bars in the graphs show the means ± SDs of 3 independent experiments. *P < .05; **P < .01.