Extended Data Fig. 2: Bone marrow acetylcholine is produced by mature B cells.

a. Flow cytometry for Chat-GFP expression in mature B cells and B cell progenitors in the bone marrow of Chat^GFP mice. b. Flow cytometry gating for Chat-GFP expression by mature B cells in the bone marrow of Chat^GFP mice and C57BL/6 mice. c. Flow cytometry plots of GFP⁺ B-2 cells in the bone marrow of Chat^GFP mice. d-h. Flow cytometry plots of GFP expression by bone marrow cells in Chat^GFP mice. i. Chat mRNA levels assessed in Chat^GFP mice by qPCR in FACS-isolated mature GFP⁺ and GFP⁻B cells, CD45⁻/Ter119⁻ niche cells, MSC, arteriolar endothelial cells (aECs) and sinusoidal EC (sEC). P = 0.0001 for Chat^GFP+ B cell group vs. Chat^GFP- B cell group. n = 11 mice per Chat^GFP+ B cell group and n = 10 mice per Chat^GFP- B cell group, two-tailed Student’s t-test. j. Intravital microscopy of the skull obtained in a Chat^GFP mouse. Endothelial cells are labeled by injecting CD31/Sca-1 and B cells by B220. Arrows indicate GFP⁺ B220⁺ B cells. The experiment was repeated three independent times with a similar result. Scale bar, 10 µm. k. Confocal microscopy of the femur in Chat^GFP mice (green). Endothelial cells are labeled by injecting CD31/ Sca-1 (blue). B220 control staining was performed using an isotype antibody (red). The experiment was performed once. Scale bar, 15 µm. l. Flow cytometry dot plots of cell cycle analysis of immature B cells in bone marrow of Chat^GFP mice. m. Quantification of immature B cells in S/M/G2 cell cycle phase. P = 0.0040. n = 3 mice per group, two-tailed Student’s t-test. n. Flow cytometry plots of Chat^GFP expression by circulating mature B cells in blood of Chat^GFP mice. o. Percentage and numbers of GFP⁺ B cells in blood of Chat^GFP mice. P = 0.0006 for percentage and P = 0.0023 for numbers of GFP⁺ B cells. Two independent experiments, n = 7 mice per group, two-tailed Mann⁻Whitney test. Data are mean ± SEM with individual values for data distribution, **P < 0.01, ***P < 0.001 and ****P < 0.0001.