Figure 3

Surface CXCR4 expression increases after treatment with endocytosis inhibitors. A) Treatment of fMSC for 24 hr with a neutralizing antibody against SDF-1 at a range of antibody concentrations. fMSC show only low level increase of CXCR4 expression (MFI calculated from flow cytometry data). B) Treatment of fMSC with the endocytosis inhibitors, blebbistatin or dynasore increases surface expression of CXCR4. Cells were treated with vehicle (0 μM) or 20–100 μM blebbistatin or dynasore for 60 min before surface expression was determined by flow cytometry (expressed as% total cells expressing surface CXCR4 ± SD). C) Kinetics of CXCR4 exocytosis in fMSC after treatment with endocytosis inhibitors. Cells were treated with 80 μM blebbistatin or dynasore then fixed and stained with anti-CXCR4 (12G5) at 0, 15, 30, 60, 90 and 120 min time points. D) MSC were incubated with vehicle or 80 μM blebbistatin or dynasore for 60 min. Fetal MSC were stained with isotype control (upper panel) or anti-CXCR4 (12G5, lower panel). (E) Inhibitor treated adult bone marrow MSC anti-CXCR4 (ab2074). Percentage of cells positive for CXCR4 expression over isotype control is indicated. Dynasore and Blebbistatin inhibit SDF-1 induced endocytosis of CXCR4 in THP-1 cells. F) In the untreated state, anti-CXCR4 antibody 12G5 detected >90% cells with surface expression of CXCR4 on THP-1 monocytic leukemia cells. G) Stimulation of THP-1 cells with 1 mg/ml of SDF-1 resulted in down-regulation of surface expression of CXCR4 while co-treatment with either blebbistatin (H) or dynasore (I) showed reduced CXCR4 endocytosis. The position of the isotype control is indicated by the gates (fluorescence intensity vs. forward scatter).