Fig. 5. p190 is required for semaphorin-induced repulsion of primary endothelial cells. (A) MDA-MB435 mammary carcinoma cells were engineered to express myc-tagged Sema3F, Sema3A, Sema4D or mock transfected. The secretion of the semaphorins in the conditioned medium as demonstrated by immunoblotting with anti-myc antibodies. (B) HUVEC cells were engineered to express siRNAs targeted to p190 transcript or to an unrelated sequence. The expression levels of p190 in cell lysates were then analysed by immunoblotting with specific antibodies. (C) siRNA-expressing HUVECs analysed as in B were grown to confluence on glass coverslips. 6x10³ MDA-MB435 tumour cells engineered to express the indicated semaphorins (as shown in A) were then seeded onto the monolayer of endothelial cells. After 48 hours of co-culture, the cells were fixed and analysed by immunofluorescence with anti-myc antibodies to reveal semaphorin-expressing cells. HUVECs were identified by GFP expression (associated with siRNA-expression vectors). Scale bar: 200 µm. In other experiments, the endothelial cell monolayers were revealed by immunocytochemistry with anti-CD31 antibodies (supplementary material Fig. S3). (D) HUVEC-free areas were identified by GFP expression or CD31 positivity (as described above) and measured by ImageQuant software. At least three independent low magnification fields were analysed, by two separate investigators, for each experimental point. The table shows average values.