Loss of Twist1 enhances Ca²⁺ level, mitochondrial activity and metabolism, and ROS level in HSCs upon genotoxic stresses. (A-B) The concentrations of intracellular Ca²⁺ (A) and mitochondrial Ca²⁺ (B) in LT-HSCs after IR stress. Representative fluorescence-activated cell sorting (FACS) plots show the fluorescence intensity of Fluo-4 (intracellular Ca²⁺) and Rhod-2 (mitochondrial Ca²⁺; n = 5). (C) Mitochondrial membrane potential of LT-HSCs determined by DilC₁(5) staining after IR (n = 5). (D) Mitochondrial membrane potential of LT-HSCs determined by TMRM staining after IR (n = 5). (E) Glucose uptake of LT-HSCs after IR (n = 5). (F) Mitochondrial mass of LT-HSCs evaluated by using MTG staining in the presence of verapamil after IR (n = 5). (G) Relative mitochondrial DNA (mtDNA) copy number of LSK cells evaluated by DNA quantitative polymerase chain reaction (PCR; n = 2). (H) Relative ROS levels of LT-HSCs after IR (n = 5). (I) Representative TEM images of mitochondrial morphology in LSK cells after 5-FU treatment (left, 10 000×; right, 20 000×). (J) Expression of Mfn2 messenger RNA based on quantitative reverse transcription PCR and MFN2 protein determined by FACS in LT-HSCs after 5-FU treatment (n = 3-5). (K) Oxygen-consumption rate (OCR) of LSK cells after 5-FU treatment (n = 4). (L) Relative ATP levels of MPPs and LT-HSCs after 5-FU treatment (n = 4). (M) Relative ROS levels of LT-HSCs after 5-FU treatment (n = 5). Data are shown as means ± standard deviation. *P < .05, **P < .01 (Student t test). MFI, mean fluorescence intensity; ns, not significant.