Figure 1.
Differentially expressed promoters in OxPhosi-resistant and OxPhosi-sensitive AML cells cocultured with MSCs are associated with reduced antileukemic efficacy of OxPhos inhibition. (A) OCI-AML3, MOLM13, and U937 cells were treated with the indicated doses of the OxPhosi IACS-010759 for 72 hours in the presence or absence of MSCs. The number of viable cells (percent of control) was determined with a Vi-Cell XR cell counter using the trypan blue exclusion method. Gray bars, cultured without MSCs; black bars, cultured with MSCs. MSCs were treated with IACS-010759 for 72 hours in single-culture conditions. (B) Adhesion of OCI-AML3 cells to cocultured MSCs. OCI-AML3 cells were cultured on MSCs for 48 hours with or without IACS-010759 (30 nM). Adherent cells were counted as described in the supplemental Materials and Methods. (C) The OCR was measured by extracellular flux assay in OCI-AML3 and MOLM13 cells treated with IACS-010759 (30 nM) with or without MSCs for 2 hours. Cocultured AML cells were separated from the MSC monolayer by careful pipetting before the extracellular flux assay. A total of 5 × 105 cells per well were used. Three technical replicates for each condition were plated. During the assay, oligomycin (Oligo), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, and antimycin A were added according to the manufacturer’s instructions as described in the supplemental Materials and Methods. Representative of the results from 3 independent Cell Mito Stress Tests, and calculated values for the baseline OCR (indicated by the arrow) are shown. Comparisons of the reductions in OCR by treatment with IACS-010759 (IACS) between the single-culture condition and MSC coculture condition are shown on the right. (D) OCR was measured by extracellular flux assay in MSCs treated with IACS-010759 (30 nM) for 2 hours. A total of 6 × 104 cells per well were used. 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 Cell Mito Stress Test results (n = 3), and calculated values for the baseline OCR (indicated by the arrow) are shown. (E-F) OCI-AML3 cells were treated with 20 nM or 30 nM IACS-010759 for 72 hours in the presence or absence of MSCs. Nonadherent OCI-AML3 cells were separated from the MSC monolayer by careful pipetting. OCI-AML3 cells that adhered to MSCs were separated from cocultured MSCs by magnetic-activated cell sorting (MACS) using anti-CD45 microbeads after trypsinization as described in panel E (supplemental Materials and Methods) or (F) separated by the transwell insert into upper and lower compartments. Apoptosis of treated cells was detected by staining with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and an anti-CD45 allophycocyanin (APC)-conjugated antibody using flow cytometry. Representative flow cytometry plots showing annexin V (x-axis) and PI (y-axis) staining are shown on the right . Error bars in the graphs show the means ± SDs of the results from 3 independent experiments. *P < .05; **P < .01.

Differentially expressed promoters in OxPhosi-resistant and OxPhosi-sensitive AML cells cocultured with MSCs are associated with reduced antileukemic efficacy of OxPhos inhibition. (A) OCI-AML3, MOLM13, and U937 cells were treated with the indicated doses of the OxPhosi IACS-010759 for 72 hours in the presence or absence of MSCs. The number of viable cells (percent of control) was determined with a Vi-Cell XR cell counter using the trypan blue exclusion method. Gray bars, cultured without MSCs; black bars, cultured with MSCs. MSCs were treated with IACS-010759 for 72 hours in single-culture conditions. (B) Adhesion of OCI-AML3 cells to cocultured MSCs. OCI-AML3 cells were cultured on MSCs for 48 hours with or without IACS-010759 (30 nM). Adherent cells were counted as described in the supplemental Materials and Methods. (C) The OCR was measured by extracellular flux assay in OCI-AML3 and MOLM13 cells treated with IACS-010759 (30 nM) with or without MSCs for 2 hours. Cocultured AML cells were separated from the MSC monolayer by careful pipetting before the extracellular flux assay. A total of 5 × 105 cells per well were used. Three technical replicates for each condition were plated. During the assay, oligomycin (Oligo), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, and antimycin A were added according to the manufacturer’s instructions as described in the supplemental Materials and Methods. Representative of the results from 3 independent Cell Mito Stress Tests, and calculated values for the baseline OCR (indicated by the arrow) are shown. Comparisons of the reductions in OCR by treatment with IACS-010759 (IACS) between the single-culture condition and MSC coculture condition are shown on the right. (D) OCR was measured by extracellular flux assay in MSCs treated with IACS-010759 (30 nM) for 2 hours. A total of 6 × 104 cells per well were used. 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 Cell Mito Stress Test results (n = 3), and calculated values for the baseline OCR (indicated by the arrow) are shown. (E-F) OCI-AML3 cells were treated with 20 nM or 30 nM IACS-010759 for 72 hours in the presence or absence of MSCs. Nonadherent OCI-AML3 cells were separated from the MSC monolayer by careful pipetting. OCI-AML3 cells that adhered to MSCs were separated from cocultured MSCs by magnetic-activated cell sorting (MACS) using anti-CD45 microbeads after trypsinization as described in panel E (supplemental Materials and Methods) or (F) separated by the transwell insert into upper and lower compartments. Apoptosis of treated cells was detected by staining with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and an anti-CD45 allophycocyanin (APC)-conjugated antibody using flow cytometry. Representative flow cytometry plots showing annexin V (x-axis) and PI (y-axis) staining are shown on the right . Error bars in the graphs show the means ± SDs of the results from 3 independent experiments. *P < .05; **P < .01.

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