Integrin expression on CD34⁺cells and integrin-dependent binding of CCN1 to CD34⁺cells. (A) Expression of integrins on CD34⁺ cells was analyzed by flow cytometry using antibodies against integrins α_M, β₂, α_V, β₃, α₆, and β₁, respectively (filled graphs). Open graphs represent isotype controls. Cells in the histograms are gated for CD34 expression. (B) 1 × 10⁵ CD34⁺ cells pretreated with 1 mM RGD peptides (RGD and GRGDSP) or left untreated were incubated with 50 ng of CCN1 for 4 hours. After centrifugation of the cells supernatants and cell pellets were subjected to Western blot analysis using antibodies against CCN1. One representative blot of 3 independent experiments is shown. (C) Immunofluorescent staining of CD34⁺ cells with an antibody against CCN1 in the absence or after preincubation with 1, 10, and 100 ng/mL CCN1 and/or with 1 mM RGD peptides (RGD and GRGDSP) for 30 minutes followed by several washing steps. Immunofluorescence was significantly increased compared with conditions without CCN1 (*P < .01) and significantly reduced by RGD-peptides compared with 100 μg/mL CCN1 (#P < .05). Data represent the mean (± SEM) immunofluorescence of 25 to 50 analyzed cells per condition. (D) Immunofluorescent staining of CD34⁺ cells in the absence (control) or after 30-minute stimulation with 1, 10, and 100 ng/mL CCN1 using antibodies against phosphorylated focal adhesion kinase (p-FAK, Ser⁷²²) or focal adhesion kinase (FAK). p-FAK-specific immunofluorescence was normalized to FAK-specific immunofluorescence. Immunofluorescence was significantly increased compared with conditions without CCN1 (*P < .01). Data represent the mean (± SEM) immunofluorescence of 25 to 50 analyzed cells per condition.