Figure 3. EphB2 Signaling Mediates Fibroblast-Induced Schwann Cell Sorting.

(A) Immunofluorescence staining for S100β (S100) and fibronectin (FBN) of SC cultured alone (SC), in direct contact with Fb (SC+Fb), in the presence of Fb conditioned medium (SC+Sol), on Fb-secreted ECM (SC+Mat) or on Fb membranes and ECM (SC+Mat+Mem). Cells were fixed 24 hr after seeding.

(B) Quantification of SC clustering in the conditions depicted in (A). For this and all later experiments, a minimum of 200 cells per coverslip was counted across randomly selected fields of view, and the percentage of SC found in clusters of increasing size was calculated. Error bars indicate the SD across repeats of each condition (n = 2–3). Shown is a representative experiment of several that gave similar results. (*** p < 0.001).

(C) Quantification of SC clustering. Samples are SC monocultures (SC), direct cocultures of SC and Fb (SC+Fb) and SC monocultures in the presence of fibroblast membrane fractions (SC+mem).

(D) Quantification of clustering in SC cultures without (SC) or with Fb (SC+Fb) pretreated with control proteins (Ctl) or soluble recombinant EphB2-Fc fusion proteins (SC EphB2-Fc).

(E) Quantification of clustering of scr siRNA-treated SC in the absence (SC Scr) or presence of Fb (SC+Fb Scr) and EphB2 siRNA-treated SC cultured in the presence of Fb (SC+Fb EphB2). Western blots show efficacy of knockdown with two independent oligos.

See also Figure S3.