Extended Data Fig. 3: Bone marrow acetylcholine preserves SLAM LSK stemness by reducing proliferation.
From: B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis
a. Bodyweight of Cd19CreChatfl/fl or their Chatfl/fl mice in steady-state. n = 8 mice per group, two-tailed Student’s t-test. Blood pressure in Cd19CreChatfl/fl or their Chatfl/fl mice in steady-state for 5 consecutive times and each individual measurement value is plotted. n = 8 mice per group, two-tailed Student’s t-test. Heart rate of Cd19CreChatfl/fl or Chatfl/fl mice. n = 8 mice per group, two-tailed Student’s t-test. b. Cell cycle of HPSCs was analyzed by flow cytomery in Cd19CreChatfl/fl or Chatfl/fl mice. Left, flow cytometry plots for cell cycle analysis of LSK and SLAM LSK. Right, cell cycle analysis of LSK and SLAM LSK by flow cytometry. SLAM LSK: P = 0.021 for G0-phase, P = 0.034 for G1-phase and P = 0.038 for S/G2/M-phase. LSK: P = 0.0002 for G0-phase, P = 0.0012 for G1-phase and P = 0.0003 for S/G2/M-phase. SLAM LSK: n = 8 mice per group, LSK: n = 7 Chatfl/fl, n = 8 Cd19CreChatfl/fl mice, two-tailed Student’s t-test. c. Limiting dilution assay. SLAM LSK from Cd19CreChatfl/fl or Chatfl/fl mice were FACS sorted and mixed with 3×105 BMNC from CD45.1 mice. Cell dilutions were transplanted into lethally irradiated CD45.1 recipients. Multilineage blood chimerism of 0.1% served as a cut-off to determine responders. d. Left table lists dilution steps, numbers of mice analyzed 4 months after transfer, and number of responders. Right table indicates HSC frequencies and 95% confidence interval (P = 0.452 for difference in frequency). e. Cd19CreChatfl/fl or Chatfl/fl mice were injected with Evans blue (20 mg/kg; i.v.) and tibias were harvested 4 hours thereafter. Vascular permeability by Evans blue quantification in the tibia. P = 0.0487. n = 7 Chatfl/fl, n = 6 Cd19CreChatfl/fl mice, two-tailed Student’s t-test. Data are mean ± SEM, n.s. P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001.
