Fig. 4: Stk11 in ECs regulates soluble stem cell factor (sScf) to drive DC differentiation.

a Deep imaging of Cx3cr1-GFP⁺ cells (green), hematopoietic progenitors (c-Kit⁺, red), and blood vessels (laminin, gray) in digitally reconstructed bone marrow (300 μm thick). Scale bar: 20 μm. Images are representative of three independent experiments. b, c Supernatant of cultured primary bone marrow ECs from WT or Stk11^ec−/− mice was subjected to soluble Scf (sScf) ELISA (b, upper) (n = 4 each group). *P = 0.005 versus WT by Student’s t-test (two-sided). Lysates of primary bone marrow ECs from WT or Stk11^ec−/− mice were analyzed by western blotting for Scf, Lkb1, and β-Actin (b, lower), and by PCR for Scf (c, upper). The serum Scf level from WT or Stk11^ec−/− mice was detected by ELISA (c, lower; 12-weeks-old, mixed-gender, WT, n = 14; Stk11^ec−/−, n = 13). *P < 0.001 versus WT by Student’s t-test (two-sided). The blots are representative results of three independent experiments. d Bone marrow cells (1 × 10⁶ cells/per well) isolated from WT or Stk11^ec−/− mice were cultured with or without c-Kit inhibitor ISCK03 (1 µM) for 6 days to generate DCs. Primary DCs were harvested from each dish by collecting non and loosely adherent cells and stained for CD11c, detected by flow cytometry. DCs were defined as CD11c⁺ cells. The colonies (red head) and megakaryocytes (green head) were stained with Giemsa. Scale bar: 1000 μm. Bar graph summarizes the numbers of DCs and megakaryocytes per cm² in culture. *P < 0.001 versus WT + vehicle by Student’s t-test (two-sided). e Bone marrow cells (1 × 10⁶ cells/per well) isolated from WT or Stk11^ec−/− mice were cultured with or without recombinant murine stem cell factor (rmScf, 50 ng/mL) for 6 days to generate DCs. Primary DCs were harvested from each dish by collecting nonadherent and loosely adherent cells and stained for CD11c, detected by flow cytometry. DCs were defined as CD11c⁺ cells. The colonies ⁽Red head) and megakaryocytes (green head) were stained with Giemsa. Scale bar: 1000 μm. Bar graph summarizes the numbers of DCs and megakaryocytes per cm² in culture. *P < 0.001 versus WT + Vehicle and ^†P < 0.001 versus Stk11^ec−/− + vehicle by Student’s t-test (two-sided). f Flow cytometry analysis of spleen/lymph node cells isolated from WT or Stk11^ec−/− mice treated with either AAV-acGFP or AAV-sScf and stained for CD11c and MHC II or CD11c and B220. Bar graph summarizes frequency of spleen and lymph node cDCs and pDCs (12-weeks-old, mixed-gender, n = 5–6 each group). *P < 0.001 versus WT and ^†P < 0.001 versus Stk11^ec−/− by nonparametric Mann–Whitney U test (two-sided). g Flow cytometry analysis of bone marrow cells isolated from WT or Stk11^ec−/− mice treated with either AAV-acGFP or AAV-sScf and stained for Lin, c-Kit, CD115, CX3CR1, and Flt3. Bar graph summarizes the ratio of MDP/CDP in bone marrow (12-weeks-old, mixed-gender, n = 5–7 each group). *P < 0.001 versus WT and ^†P < 0.001 versus Stk11^ec−/− by nonparametric Mann–Whitney U test (two-sided).