Figure 1.
HSC homeostasis is impaired in Twist1-deficient mice. (A) Relative expression levels of Twist1 in different hematopoietic cell subsets were evaluated by quantitative polymerase chain reaction. Each subset was compared with BM for statistical analysis (n = 3). LT-HSC: Flk2−CD150+CD48−LSK; ST-HSC: Flk2−CD150−CD48−LSK; multipotent progenitor 2 (MPP2): Flk2−CD150+CD48+LSK; MPP3: Flk2−CD150−CD48+LSK; MPP4: Flk2+CD150−CD48+LSK; common myeloid (My) progenitor (CMP): CD34+CD16/32−LK (Lineage−cKit+Sca1−); granulocyte/monocyte progenitor (GMP): CD34+CD16/32+LK; megakaryocyte/erythrocyte progenitor (MEP): CD34−CD16/32−LK; CLP: CD127+cKitlowSca1low Lineage−. (B) Relative expression levels of Twist1 messenger RNA (mRNA) in CD34−Flk2− LT-HSCs in G0, G1, and S/G2/M phases (n = 3). (C) LT-HSCs, ST-HSCs, and MPP2-4 cells were isolated from mice before and at 12 hours after irradiation (IR; 5 Gy). Twist1 mRNA was detected, and the fold change of Twist1 expression was normalized to the 0-Gy group (n = 3). (D) Induced expression of Twist1 mRNA in LT-HSCs, ST-HSCs, and MPP2-4 cells 10 days after 5-fluorouracil (5-FU) treatment. Fold change of Twist1 expression was normalized to the no-treatment group (n = 3). (E) Frequencies of indicated populations in BM cells from control (Ctrl) and cKO mice at 4 weeks after the last polyinosinic:polycytidilic acid injection and day 12 after IR (right) and representative fluorescence-activated cell sorting (FACS) plots (left; n = 5). (F) Percentages of CD150highLSKFlk2−CD48− and CD150lowLSKFlk2−CD48− cells in BM cells from Ctrl and cKO mice at steady state and day 12 after IR (n = 3-5). (G) Apoptosis in BM CD150low HSCs under steady-state conditions (n = 4). (H) Cell-cycle analysis of LT-HSCs at basal and day 12 post-IR (right) and representative FACS plots (left; n = 5). (I) Proliferation analysis of LT-HSCs at basal and day 12 post-IR (n = 4). (J-K) Frequencies of CMPs, GMPs, and MEPs in BM cells at steady state and day 12 after IR (K) and representative FACS plots (J) (n = 5). (L) Frequencies of common lymphoid (Ly) progenitors (CLPs) in BM under steady-state conditions and after IR (n = 5). (M) Lineage distribution in BM assessed by FACS at steady state (n = 5). (N) Absolute number per liter of white blood cells (WBCs), neutrophils, and monocytes in peripheral blood (PB) from Ctrl and cKO mice under steady-state conditions and after IR (n = 4-5). Data are represented as means ± standard deviation. *P < .05, **P < .01, ***P < .001 (Student t test). ns, not significant.

HSC homeostasis is impaired in Twist1-deficient mice. (A) Relative expression levels of Twist1 in different hematopoietic cell subsets were evaluated by quantitative polymerase chain reaction. Each subset was compared with BM for statistical analysis (n = 3). LT-HSC: Flk2−CD150+CD48−LSK; ST-HSC: Flk2−CD150−CD48−LSK; multipotent progenitor 2 (MPP2): Flk2−CD150+CD48+LSK; MPP3: Flk2−CD150−CD48+LSK; MPP4: Flk2+CD150−CD48+LSK; common myeloid (My) progenitor (CMP): CD34+CD16/32−LK (Lineage−cKit+Sca1−); granulocyte/monocyte progenitor (GMP): CD34+CD16/32+LK; megakaryocyte/erythrocyte progenitor (MEP): CD34−CD16/32−LK; CLP: CD127+cKitlowSca1low Lineage−. (B) Relative expression levels of Twist1 messenger RNA (mRNA) in CD34−Flk2− LT-HSCs in G0, G1, and S/G2/M phases (n = 3). (C) LT-HSCs, ST-HSCs, and MPP2-4 cells were isolated from mice before and at 12 hours after irradiation (IR; 5 Gy). Twist1 mRNA was detected, and the fold change of Twist1 expression was normalized to the 0-Gy group (n = 3). (D) Induced expression of Twist1 mRNA in LT-HSCs, ST-HSCs, and MPP2-4 cells 10 days after 5-fluorouracil (5-FU) treatment. Fold change of Twist1 expression was normalized to the no-treatment group (n = 3). (E) Frequencies of indicated populations in BM cells from control (Ctrl) and cKO mice at 4 weeks after the last polyinosinic:polycytidilic acid injection and day 12 after IR (right) and representative fluorescence-activated cell sorting (FACS) plots (left; n = 5). (F) Percentages of CD150highLSKFlk2−CD48− and CD150lowLSKFlk2−CD48− cells in BM cells from Ctrl and cKO mice at steady state and day 12 after IR (n = 3-5). (G) Apoptosis in BM CD150low HSCs under steady-state conditions (n = 4). (H) Cell-cycle analysis of LT-HSCs at basal and day 12 post-IR (right) and representative FACS plots (left; n = 5). (I) Proliferation analysis of LT-HSCs at basal and day 12 post-IR (n = 4). (J-K) Frequencies of CMPs, GMPs, and MEPs in BM cells at steady state and day 12 after IR (K) and representative FACS plots (J) (n = 5). (L) Frequencies of common lymphoid (Ly) progenitors (CLPs) in BM under steady-state conditions and after IR (n = 5). (M) Lineage distribution in BM assessed by FACS at steady state (n = 5). (N) Absolute number per liter of white blood cells (WBCs), neutrophils, and monocytes in peripheral blood (PB) from Ctrl and cKO mice under steady-state conditions and after IR (n = 4-5). Data are represented as means ± standard deviation. *P < .05, **P < .01, ***P < .001 (Student t test). ns, not significant.

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