Figure 3.

Effect of inhibiting Ryk on Wnt5a-mediated proliferation of LSK cells. LSK and LSK, CD150⁺, CD48⁻ cells were cultured as described under Materials and Methods section. (A): Average fold-expansion of LSK (left) and LSK, CD150⁺, CD48⁻ cells (right) after 4 days in culture with rWnt5a and anti-Ryk antibody (α-Ryk) (n=3). (B): Representative flow cytometry analysis of cell cycle status of LSK cells cultured for 3 days under control conditions (top), with rWnt5a (middle), and with rWnt5a+α-Ryk (bottom). Percentages of cells in G₀, G₁, and S/G₂/M are displayed in their respective gates. (C): Average percentage of LSK cells in S/G₂/M phases of the cell cycle after 3 days in culture (n=3). (D): Average percentage of LSK cells in G₀ after 3 days in culture (n=3). (E): Average colony forming units-granulocyte, macrophage frequency in LSK cells after 4 days in culture (n=3). For all panels, error bars represent SD and p values were determined using paired t test. Abbreviations: APC, allophycocyanin; LSK, lin⁻, Sca-1⁺, c-kit⁺; Ryk, related to receptor tyrosine kinase; rWnt5a, recombinant Wnt5a.