Loss of Sel1L leads to HSC hyperproliferation and activation. (A) Two weeks after pIpC injection, Mx1-cre⁺; Sel1L^fl/fl (fl/fl) or control (^+/+) mice were injected with BrdU (200 mg/kg body mass; IP) and then placed on drinking water containing BrdU (1 mg/mL) for 24 hours. Representative FACS plots of gating strategy (left) and summary of BrdU incorporation in HSCs (right) are shown. (B) Six- to 8-week-old Vav1-cre⁺; Sel1l^fl/fl(fl/fl) and Vav1-cre⁻; Sel1l^fl/+ or Vav1-cre⁻; Sel1l^fl/fl (^+/+) mice were injected with BrdU (200 mg/kg body mass; IP) and then placed on drinking water containing BrdU (1 mg/mL) for 24 hours. Summary of BrdU incorporation in HSCs are shown. (C) Representative FACS plots with gating strategy (left), and summary of H2B-GFP retention (right) in HSCs after 12 weeks of off-doxycycline chase. Two weeks after pIpC injection, Mx1-cre⁺; Sel1L^fl/fl; Col1A1-H2B–GFP^+/+; Rosa26-M2-rtTA^+/+ (fl/fl) or control (^+/+) mice were placed on doxycycline water (2 g/L). After 6 weeks’ labeling, doxycycline water was removed and GFP level in HSCs was analyzed after 12 weeks “off-label” chase. Two weeks after pIpC injection, Mx1-cre⁺; Sel1L^fl/fl (fl/fl) or control (^+/+) mice were analyzed for mitochondrial membrane potential (D, by TMRM), mitochondrial mass (E, by Mito tacker green), and (F) ratio of mitochondrial membrane potential to mass. (D-F) Mean fluorescence intensity (MFI) were normalized to mean value on the day of measurement, and P values were determined by 2-sided Wilcoxon rank sum test. (G-H) Apoptosis was measured by Annexin-V staining (G, in SLAM HSCs) or Caspase 3/7 activity (H, in CD48⁻LSK) on Mx1-cre⁺; Sel1L^fl/fl (fl/fl) or control (^+/+) mice 2 weeks after pIpC. Data represent mean ± SD. Two-sided Student t test was used for statistical analysis unless specified.