JCS02590 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Downregulation of Rho-GTP in response to PlxnA1 signalling. NIH-3T3 fibroblasts expressing either PlxnB1, or the chimeric receptor PlxnB1/A1 (carrying the cytoplasmic domain of PlxnA1) were treated with the ligand Sema4D (5 nM) for the indicated times. Rho-GTP levels were measured in non-serum-starved NIH-3T3 fibroblasts by pull-down with rhotekin-RBD-coated beads, followed by immunoblotting with anti-Rho antibodies. Total Rho and plexin expression in cell lysates is shown below.

• Supplemental Figure 2 - Fig. S2. The spreading of p190^–/– fibroblasts on fibronectin is insensitive to plexin signalling. Fibroblasts derived from p190^+/+ or p190^–/– mice were engineered to express PlxnB1 and their spreading on fibronectin-coated wells was tested after 30 minutes incubation in the presence or absence of 10 nM Sema4D. Cells were then fixed and stained with Crystal Violet. Representative fields are shown on the left. Scale bar: 40 mm. Plexin activation inhibited lamellipodia extension and spreading of wild-type fibroblasts, but was ineffective on p190-deficient cells. The fraction of spread cells was determined for each condition (by counting at least 100 cells in multiple independent fields) and it is shown in the bar chart on the right.

• Supplemental Figure 3 - Fig. S3. Endothelial cell repulsion by semaphorin-expressing cells. Shown are the results of similar experiments to those shown in Fig. 5: endothelial cells are revealed here by immunocytochemistry with anti-CD31 antibody, followed by incubation with peroxidase-coupled secondary antibody. All images were taken using 103 magnification. MDA-MB435 mammary carcinoma cells were found in empty spaces within the monolayer, however, being poorly adherent, they had been largely removed during the immunostaining procedure.