Blockade of ICAM-1 and actin polymerization inhibits mitochondrial transfer and increases cytotoxic effects of OxPhos inhibition. (A-B) PDHA1-GFP–transfected OCI-AML cells were cocultured with PDHA1-dsRed–transfected MSCs with or without IACS-010759 (30 nM) and/or an ICAM-1 neutralizing antibody (Ab) (25 μg/mL) (left) or cytochalasin D (Cyto D) (350 nM) (right). (A) To quantitatively determine the rate of mitochondrial transfer, the number of GFP and dsRed dual-positive recipient cells per 100 GFP⁺ cells was counted at ×40 magnification by live-cell imaging with confocal microscopy. Laser scanning was used to obtain images under a confocal microscope. To quantitatively determine the rate of mitochondrial transfer, OCI-AML3 cells cocultured with MSCs were separated from the MSC monolayer by careful pipetting, and the GFP and dsRed dual-positive recipient cells per 100 GFP⁺ cells were counted at ×40 magnification by live-cell imaging with confocal microscopy (right panel). Laser scanning was used to obtain images under a confocal microscope. The means ± SDs of the results from 5 independent experiments. (B) The percentage of dead cells was determined by the trypan blue exclusion method. (C) The OCR was measured by extracellular flux assay in OCI-AML cells cocultured with MSCs with or without IACS-010759 (20 nM) and/or an ICAM-1 neutralizing antibody (25 μg/mL). Cocultured OCI-AML3 cells were separated from the MSC monolayer before the extracellular flux assay. A total of 5 × 10⁵ AML cells were added per well. Three technical replicates for each condition were plated. During the assay, oligomycin, FCCP, rotenone, and antimycin A were added according to the manufacturer’s instructions. Representative of the results from 3 independent Cell Mito Stress Tests, and calculated values for the baseline OCR (indicated by the arrow) are shown. Error bars in the graphs show the means ± SDs of the results from 3 independent experiments. Two-way analysis of variance *P < .05; **P < .01. Cont, control.