ddC depletes mtDNA and its encoded proteins, inhibits oxidative phosphorylation, and induces preferential antileukemic effects. (A) TEX and OCI-AML2 cells were treated with ddC for 3 and 6 days. Relative mtDNA content was assessed by qRT-PCR as described in “Materials and methods.” Mean ± SD; n = 3. (B) Effect of ddC treatment on protein levels of mitochondrial COX I (mt-COX I), mt-COXII, nuclear COX IV (nu-COX IV), and β-tubulin in whole-cell extracts of TEX and OCI-AML2 cells. The immunoblot from a representative experiment is shown. (C-D) Basal OCR was assessed in TEX and OCI-AML2 cells following ddC treatment of 3 and 6 days, respectively, using the Seahorse XF96 Metabolic Flux Assay. Mean ± SD; n = 3. (E-F) Effect of ddC on cell viability and proliferation in TEX and OCI-AML2 cells. Cell viability was assessed by trypan blue exclusion staining. Mean ± SEM; n = 3. (G) Primary leukemia and normal hematopoietic progenitor cells (G-CSF–mobilized PBSCs) were treated with 2 µM ddC for 6 days. mtDNA content was assessed by qRT-PCR. Leukemia samples C1-C9 were used for analysis. (H) Normal PBSCs were treated with 2 µM ddC for 6 days and sorted for the CD34⁺ subpopulation using immunomagnetic selection. mtDNA content was assessed in CD34⁺ population by qRT-PCR. (I) Cell viability was assessed by trypan blue exclusion staining in primary AML cells and Cyquant DNA staining for PBSCs from panel G. Dotted line indicates the cutoff to stratify samples as ddC-sensitive or ddC-resistant. (J) Normal PBSCs were treated with 2 µM ddC for 6 days and cell viability was assessed by propidium iodide (PI) staining in CD34⁺ subpopulation by flow cytometry. For all experiments, *P < .05, **P < .01, ***P < .001, and ****P < .0001 using the Bonferroni posttest after 1-way ANOVA. The Student t test was applied to panels G-J. DMSO, dimethyl sulfoxide.