Figure 3. Identification of a Notch3⁺ sub‐population within the adipogenic lineage cells.

• A
  t‐SNE plots showing the original (left) and re‐clustered (right) adipogenic lineage cells by integrated analysis of homeostatic and stress conditions. Top 15 PCs were chosen for the clustering.
• B
  Dot plots showing the gene expression patterns of selected genes in each cluster.
• C
  Stacked bar charts showing the cell cycle status in all clusters.
• D
  Flow cytometry analysis of BrdU incorporation in uncultured Lepr‐Cre⁺Notch3^+/− BMSCs (n = 4 independent experiments). Eight‐week‐old Lepr‐Cre; tdTomato mice were given a single intraperitoneal injection of BrdU (100 mg/kg body mass) and maintained on 0.5 mg/ml of BrdU in the drinking water for 14 days.
• E
  Immunofluorescent images of the distal femur in 8‐week‐old Lepr‐Cre; tdTomato mice. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Sinusoid (a), arteriole (b), artery (c), and avascular bone marrow (BM, d) regions are shown. Arrowheads indicated Notch3⁺ tdTomato⁺ adventitial cells closely associated with ECs. Arrows indicated Notch3⁻ tdTomato⁺ reticular cells in the bone marrow that were not associated with ECs. Scale bars are 10 µm.
• F
  Localization quantification of Lepr‐Cre⁺Notch3⁺ cells relative to bone marrow ECs (n = 5 biological replicates from three independent experiments). Cells within 5 µm diameter of ECs were considered adjacent.
• G
  Graphical illustration of the 3D co‐culture system.
• H
  Volcano plot showing DEGs in the bulk RNA‐seq analysis (n = 3 biological replicates). Green dots represent down‐regulated genes. Red dots represent up‐regulated genes.
• I
  qPCR analysis of Notch3 and Cdk6 expression (n = 3 independent experiments).
• J
  Flow cytometry analysis of BrdU incorporation in Lepr‐Cre⁺Notch3^+/− BMSCs after 3D co‐cultured with bone marrow ECs (n = 3 independent experiments). BrdU was administered at a final concentration of 10 µM from culture day 1 to day 5.
• K, L
  GO terms (K) and KEGG pathways (L) enriched in up‐regulated genes.
• M
  Representative confocal images of BMSCs 3D co‐cultured with bone marrow ECs. Z‐stack projection is shown. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Arrowheads indicated Notch3⁺tdTomato⁺ adventitial cells. Arrows indicated bone marrow ECs. Scale bar is 50 µm.
• N
  Localization quantification of Lepr‐Cre⁺Notch3⁺ cells relative to bone marrow ECs after 3D co‐culture (n = 3 biological replicates from three independent experiments). Cells within 25 µm diameter of ECs were considered adjacent.
• O
  Heatmap showing the average expression levels (column‐wide Z score) of secreted factors with clustering.

Data information: All data represented Mean ± SD. The statistical significance of differences was analyzed by two‐tailed unpaired Student’s t‐test. *P < 0.05, ***P < 0.001.