Figure 5. Increase in integrin signaling mediates adhesion of IkE5^Δ/Δ pre-B cells to a stromal niche.

a, Pathway analysis of genes upregulated in IkE5^Δ/Δ relative to WT large pre-B cells. Analysis was performed with a signature of upregulated genes shared by ex vivo mutant pre-B cells prior to and after limited stromal expansion. Pathways enriched for integrins and integrin signaling effectors are highlighted in red. b, Upregulated expression of components of the integrin-actin cytoskeleton pathway in primary and cultured WT and IkE5^Δ/Δ pre-B cells as defined in Figs. 1 and 3. Hierarchical clustering of normalized gene expression values across different conditions is shown. c, Cell surface expression of integrins α5, β6, and activated β1 in ex vivo sorted and in vitro cultures of large pre-B cells. MFI for integrin staining is provided. d-f, Increase in FAK activation measured by flow cytometry, immunoblot and confocal microscopy. d, Flow cytometric analysis of p-FAK expression in ex-vivo and in vitro cultured large pre-B cells. MFI for p-FAK is indicated. e, Confocal immunofluorescence microscopy detection of activated p-FAK (red channel), GFP-expressing OP9 stroma (green channel), and nuclei (DAPI, blue channel). Scale bar, 25 μm. f, Immunoblot analysis of total FAK and activated p-FAK, with Btk as a loading control as in Fig. 4a. g, Adhesion of WT and IkE5^Δ/Δ adherent pre-B cells to fibronectin-coated plates (left panel) in the presence of the fibronectin-derived RGD peptide or the RGE mutant variant (right panel). Asterisks denote significant differences in adhesion between mutant pre-B cells in the presence or absence of RGD or RGE peptides (n=3; *P < 0.05, two-tailed Student's t-test). h, Chemotaxis of WT (circle) and IkE5^Δ/Δ (square) pre-B cells measured in a transwell migration assay in the presence of SDF1. The mean percentage of cells recovered at the bottom of the well in two independent studies is shown.