Fig 2: Lkb1-dependent Ptbp1 phosphorylation promotes Scf exon 6 retention to induce the formation of soluble stem cell factor (sScf) and drives DC differentiation.a Reverse transcription-polymerase chain reaction (RT-PCR) analysis of soluble Scf (including exon 6) to membrane-bound Scf (lacks exon 6) in control or target-gene-silenced primary mouse bone marrow ECs. *P = 0.02 versus Control si by nonparametric Mann–Whitney U test (two-sided). b RNA-binding protein was pull down using oligo-dT beads in indicated conditions and subjected to western blotting of Ptbp1 binding. *P = 0.02 versus vehicle by nonparametric Mann–Whitney U test (two-sided). c RT-PCR analysis of soluble Scf/membrane-bound Scf ratio in vehicle or peptide nucleic acid (PNA)-treated bone marrow ECs. *P = 0.03 versus vehicle by nonparametric Mann–Whitney U test (two-sided). d RNA-binding protein (RBP) was pulled down using oligo-dT beads in WT (vehicle-treated) or PNA competition assay and subjected to western blotting of Ptbp1 binding. FT, flow-through; rb-Ptbp1, RNA binding Ptbp1; WCL, whole-cell lysate. *P = 0.004 versus Control si by nonparametric Mann–Whitney U test (two-sided). e Proteins in in vitro kinase assay reaction mixture were separated on a Phos-tagTM gel and analyzed by western blotting with the anti-Ptbp1 antibody. # marked the phosphorylated band. Blots are representative of three independent experiments. f Endothelial cells (ECs) were transfected with His-Ptbp1wt and mutant plasmid and whole-cell lysates were analyzed by western blotting for anti-His after separation in Phos-tagTM gel. # marked the phosphorylated band. Blots are representative of three independent experiments. g PCR analysis was carried out to detect soluble Scf/membrane-bound Scf ratio in BAECs transfected with Ptbp1wt and mutant plasmid. *P = 0.003; †P = 0.007 by nonparametric Mann–Whitney U test (two-sided). h Flow cytometry analysis of bone marrow cells isolated from WT or Stk11ec−/− mice treated with either AAV-acGFP or AAV-Ptbp1T138E 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 = 5–6 each group). *P < 0.001 versus WT and †P < 0.001 versus Stk11ec−/− by nonparametric Mann–Whitney U test (two-sided). i Flow cytometry analysis of spleen/lymph node cells isolated from WT or Stk11ec−/− mice treated with either AAV-acGFP or AAV-Ptbp1T138E and stained for CD11c and MHC II or CD11c and B220. Bar graph summarizes the frequency of cDCs and pDCs in spleen, and lymph node (12-weeks-old, mixed-gender, n = 5–6 each group). *P < 0.001 versus WT and †P < 0.001 versus Stk11ec−/− by nonparametric Mann–Whitney U test (two-sided).

Fig 3: 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 Stk11ec−/− 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 Stk11ec−/− 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 Stk11ec−/− mice was detected by ELISA (c, lower; 12-weeks-old, mixed-gender, WT, n = 14; Stk11ec−/−, 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 Stk11ec−/− 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 Stk11ec−/− 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 Stk11ec−/− + vehicle by Student’s t-test (two-sided). f Flow cytometry analysis of spleen/lymph node cells isolated from WT or Stk11ec−/− 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 Stk11ec−/− by nonparametric Mann–Whitney U test (two-sided). g Flow cytometry analysis of bone marrow cells isolated from WT or Stk11ec−/− 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 Stk11ec−/− by nonparametric Mann–Whitney U test (two-sided).