Journal: The Journal of Clinical Investigation
Article Title: Sinusoidal ephrin receptor EPHB4 controls hematopoietic progenitor cell mobilization from bone marrow
doi: 10.1172/JCI87848
Figure Lengend Snippet: (A) EPHB1–4 and ephrin B2 in lysates of endothelial (BMEC and MS-1) and stromal (MS-5) mouse cell lines by immunoblotting; membrane reprobed for β-actin. (B) WT or K647R mutant EPHB4 expression; EPHB4 silencing with sh#1 and sh#2 in BMECs; PGK vector was used as a control. Immunoblotting of cell lysates. (C–H) Transendothelial migration of bone marrow hematopoietic cells from G-CSF–treated or control mice through: control (PGK), EPHB4-silenced (sh#1 and sh#2), WT EPHB4– (EPHB4), or EPHB4 K647R– (K647R) expressing BMECs; gelatin-coated Transwells were controls. The proportion of monocytes, neutrophils, Lin–c-Kit+Sca-1+ cells, T cells, and B cells in the cell population loaded into the upper chamber and recovered in the bottom chamber was measured by flow cytometry. Results show % cells (of total loaded) recovered after transmigration (median of 3–4 independent experiments; red lines). (I) Number of colonies in methylcellulose generated by 100 μl suspension of transmigrated cells (n = 3 experiments, each performed in triplicate). (J) BMEC monolayer permeability to Texas red–dextran; the results (% fluorescence/total loaded) reflect means of individual measurements (n = 8). (K) Adherence of tdTomato+ bone marrow Lin– cells to BMEC monolayers; the results (% cells/total loaded) reflect means of individual measurements (n = 8). (L) Distribution of EPHB4 (green) in endomucin+ (red) sinusoidal endothelium showing EPHB4 clustering toward the hematopoietic cells and away from the sinusoidal lumen. Nuclei: DAPI (blue). Boxed area is magnified on the right. Scale bars: 10 μm. Statistical significance by 1-way ANOVA with Dunnett’s multiple comparison test: *P < 0.05; **P < 0.01; ***P < 0.001.
Article Snippet: The mouse bone marrow stromal cell line MS-5 (Creative Bioarray), the mouse melanoma cell line B16F10 (ATCC), and the mouse lung carcinoma LLC1 (ATCC) were cultured in high-glucose DMEM with 10% FBS.
Techniques: Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Migration, Flow Cytometry, Transmigration Assay, Generated, Permeability, Fluorescence
