Mitochondrial dysfunction and ROS overproduction in phenotypically defined STAT3^−/− mouse HSCs and HPCs. (A) Graphic representation and dot-plot of mouse BM Lin⁻ cells and c-kit and Sca1 expression performed as in Figure 2A. Shown are the 3 subpopulations that were gated and evaluated for mitochondrial properties and ROS levels. R1 and R2 are further subdivided into 2 discrete subpopulations based on size and CD34 surface expression (see Figure 2A). The small dot-plot in the top right part of panel A is the same data, but “de-cluttered” to more easily see the overall pattern. (B) Quantitative comparison of average MitoTracker Green staining per cell (a measure of mitochondrial mass per cell) on gated subpopulations described in panel A. Also shown is a statistical comparison of results from 3 WT and STAT3^−/− pairs. (C) Staining of the indicated subpopulations with JC-1, a probe that measures mitochondrial membrane potential (δ ψ-m; ΔΨ_m), a measure of mitochondrial activity. Also shown is a quantitative and statistical analysis comparing ΔΨ_m in the 4 defined subpopulations (panel C), as well as a comparison of “activity/mass ratio” (panel D) for STAT3^−/− and WT mouse cells from the same animals used for panel B. This ratio is a measure of mitochondrial activity per cell that is normalized for mitochondrial mass (derived from data in panels B and C) and reflects the average mitochondrial membrane potential per cell irrespective of mitochondrial mass (ie, a kind of “specific” mitochondrial activity measurement). (E) Analysis of average ROS levels per cell in the indicated subpopulations in a separate experiment using 3 different WT/STAT3^−/− pairs. Error bars in panels B through E are SD of mean values and statistical significance. *P < .05 by 2-tailed Student t test. (F) OCR (obtained using a Seahorse XF96 extracellular flux analyzer/respirometer) from identical numbers of splenocytes/well from STAT3^−/− or WT from 2 separate experiments each with 1 WT and 1 STAT3^−/− mouse. Basal OCR and OCR after oligomycin-A treatment (an ATPase inhibitor) are shown. WT indicates littermate control cells; −/−, STAT3^−/− cells. Data shown are the average OCR measured from 24 wells of each type of cell where the OCR (derived from slope of pM of O₂ consumed per minute) was measured 16 times in each well simultaneously over a period of 2 hours until the OCRs were stabilized. The last stabilized average OCR measurement is presented.