Extended Data Fig. 7: Chrna7+ bone marrow stromal cells.
From: B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis
a. Dot plot of signature gene expression in Chrna7+ bone marrow cell clusters assessed by single cell RNA sequencing. Chrna7+ bone marrow stromal cells were pooled post-hoc from Chatfl/fl and Cd19Cre Chatfl/fl steady-state mice (15,590 non-hematopoietic cells; n = 8 mice). Color indicates z-score of average gene expression (red, high; blue, low), circle size indicates percent of cells expressing the gene. b. Cd19CreChatfl/fl mice or their Chatfl/fl littermates were treated with AMD3100 (5 mg/kg; s.c.) or vehicle and blood was collected 4 h after treatment for colony forming unit (c.f.u.) and flow cytometry analysis. Circulating c.f.u. assay expressed as fold change over vehicle-treated mice of same genotype. P = 0.0047. n = 7 mice per Chatfl/fl group and n = 6 per Cd19CreChatfl/fl group, two-tailed Mann-Whitney test. c. Blood leukocytes expressed as fold change over vehicle-treated mice of same genotype. P = 0.0024. n = 7 mice per Chatfl/fl group and n = 6 per Cd19CreChatfl/fl group, two-tailed Mann-Whitney test. d. Bone marrow supernatant was harvested from naive Cd19CreChatfl/fl mice or their Chatfl/fl littermates and Tnfα protein was analyzed by ELISA. P = 0.0024. n = 7 mice per Chatfl/fl group and n = 6 per Cd19CreChatfl/fl group, two-tailed Mann-Whitney test. e. Left, flow cytometry plots for gating of CD31+ endothelial cells (ECs) and LepR+ MSCs in the bone marrow. Right, flow cytometry histogram plots of Chrna7 expression on ECs (left) and LepR+ MSCs (right). Numbers in the histogram represent the mean fluorescence intensity of 3 mice per group. Bar graphs represent the percentage of Chrna7+ cells among ECs or LepR+ MSCs. Gates were set using an Chrna7 isotype control antibody. P = 0.0076 for LepR+ MSCs. n = 3 mice per group, two-tailed Student’s t test. Data are mean ± SEM with individual values for data distribution, *P < 0.05 and **P < 0.01.
