Ruxolitinib targets inflammation-induced JAK/STAT signaling in leukemia microenvironment and ROS production. (A) JAK1/2, STAT1/3/5a/5b gene expression analysis in sorted bone marrow-derived primary human mesenchymal stem and progenitor cells after coculture with primary human AML cells (n = 7) or healthy controls (n = 2) for 4 days by real-time polymerase chain reaction (PCR) (each primary sample was tested in triplicate). (B) Left, representative FACS plot of CD271⁺CD146⁺ primary human bone marrow mesenchymal stem and progenitor cells, gated on CD45^–CD235^–CD31^– cells. Right, JAK2 gene expression analysis in sorted bone marrow mesenchymal stem and progenitor cells from AML patients or nonleukemic controls at first diagnosis by real-time PCR (n = 3, data normalized to control). (C) Confocal images from primary human mesenchymal stem and progenitor cells after coculture with nonleukemic mononuclear cells (control) or primary human AML cells stained for vimentin and pSTAT3. The scale bars, 50 µm. (D) primary human AML cells (n = 7) and controls (n = 2) were cocultured with bone marrow–derived primary human mesenchymal stem and progenitor cells. MFI of pSTAT3 and pSTAT5 of stromal cells assessed by BD Phosflow technology (each sample was tested in duplicate). (E-F) Intracellular ROS production detected by measuring 2′,7′-dichlorofluorescin diacetate (DCFDA) in primary human AML cells in mono- or coculture with HS-5 stromal cells. (E) Exemplary FACS plot showing the MFI of DCFDA in 1 DMSO-treated AML patient sample mono- or cocultured with HS-5 stromal cells. (F) Intracellular ROS production in mono- or cocultured primary human AML cells after treatment with 2 µM ruxolitinib for 4 days (n = 7-9 different primary AML samples; each primary sample was tested in triplicate; connecting lines represent 1 patient sample). (G) Mitochondrial superoxide (MitoSOX) production in mono- or cocultured primary human AML cells after treatment with 2 µM ruxolitinib or vehicle using MitoSOX Red reagent (n = 6 different primary AML samples; each primary sample was tested in triplicate, connecting lines represent 1 patient sample). (H) Intracellular ROS production was detected by measuring DCFDA in mono- or cocultured primary human AML cells treated with 0.5 µM diphenyleneiodonium (DPI) or vehicle for 2 days. Left, representative FACS plot showing the MFI of DCFDA in 1 AML patient sample treated with DPI or vehicle. Right, quantification of intracellular ROS production in primary human AML cells in mono- or coculture with HS-5 stromal cells (n = 4-6 different primary AML samples; each primary sample was tested in triplicate; connecting lines represent 1 patient sample). (I) Primary human AML cells were either mono- or cocultured with HS5 cells and treated with 0.5 µM DPI or vehicle for 2 days. Absolute numbers of CD45⁺ cells were normalized to monocultured control (n = 4-6 different AML donors; each primary sample was tested in triplicate). Data are shown as mean ± SEM. See also supplemental Figure 4. *P < .05; **P < .01 determined by Student t test, Wilcoxon signed-rank test, or Mann-Whitney U test.