Transient nSMase-2 inhibition activates an ROS-driven ISR in human and murine HSPCs. Representative fluorescence intensity of human CD34⁺ cells and relative FCs in mean fluorescence intensity (MFI) of human CD34⁺ cells (blue histograms) and murine HSPCs (green histograms) after GW4869 treatment: ROS by DCFDA/DCF measurement (A), mitochondrial ROS measurement by MitoSOX assay (B), mitochondrial mass measurement by MitoTracker analysis (C), and 2-NBDG glucose analog uptake measurement (D). Representative immunoblots (E) and quantitative analysis of phosphorylated eIF2α and total ATF4 protein levels in murine HSPC^GW (F), equal protein loaded. Relative gene expression of Atf4 (G), Chop (H), and Redd1 (I) in murine HSPC^GW. (J) Relative gene expression of Atf4 across murine HSPC^GW subpopulations. Dotted lines represent normalized averages of DMSO-treated cell controls. ST, short-term time point (24 hours after GW4869 initiation). DEL, delayed time point (5 days after GW4869 withdrawal). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Refer to supplemental Figure 7.