Dual treatment with 3PO and ruxolitinib induces cell-proliferation arrest and apoptosis in human cell lines expressing VF by altering redox homeostasis. (A) Apoptosis rate was determined by percentage of annexin V⁺ cells in indicated human myeloid leukemia cells after treatment with 3PO and/or ruxolitinib for 48 hours (n = 3 independent experiments). (B) Cell proliferation was determined by percentage of Ki67⁺ cells in indicated human myeloid leukemia cells in the presence of 3PO and/or ruxolitinib for 48 hours (n = 3 independent experiments). (C) Measurements of extracellular acidification rate (ECAR), indicative of glycolytic rates, in SET2, HEL UKE1, and K562 cells after treatment with 3PO and/or ruxolitinib for 12 to 16 hours (n = 3 independent experiments). (D) Measurements of OCR, indicative of mitochondrial oxidative phosphorylation, in indicated cells after treatment with 3PO and/or ruxolitinib for 12 to 16 hours (n = 3 independent experiments). (E) Total reactive oxygen species (ROS) levels in SET2 cells after treatment with 3PO and/or ruxolitinib for 24 hours. Cells were pretreated with 1.5 mM of N-acetyl-cysteine (NAC) for 6 hours. Data were normalized to vehicle-treated control (n = 3 independent experiments). (F-G) Apoptosis rate (F) and cell proliferation (G) were determined in SET2 cells treated with 3PO and/or ruxolitinib and/or NAC for 48 hours. Cells were pretreated with NAC for 6 hours. Normalized values to vehicle-treated controls are shown. (H-I) Ratio of glutathione (GSH)/glutathione disulfide (GSSG) (H) and NADPH levels (I) in SET2 cells treated with 3PO and/or ruxolitinib for 12 hours. Cells were pretreated with NAC for 6 hours. Normalized values to vehicle-treated controls are shown. All data are presented as mean ± standard error of the mean. Two-way analyses of variance with subsequent Bonferroni posttests were used. See also supplemental Figure 7. *P < .05, **P < .01, ***P < .001. MFI, mean fluorescence intensity.