Figure 5. Effect of Cyr61 suppression or antibody neutralization on breast cancer cell motility properties.

A. MDA-MB-231 were transiently transfected with siRNA-Cyr61 for 72 h and, Cyr61 expression determined from total cell extracts by immunoblotting. Membranes were reblotted with anti-α-tubulin for loading control. Results are representative of three independent experiments in triplicate. B. For cell invasion through Matrigel-coated inserts, 48 h after siRNA-Cyr61 or siRNA-Control transfected cells were loaded onto Matrigel. After 22 h, invaded cells were fixed, stained with DAPI and counted by fluorescence microcopy. The number of invaded cells per insert is shown and represents average ± SD of three experiments performed in triplicate (**p < 0.01). C. For transendothelial migration, cells were seeded onto the HUVEC monolayer, and 48 h after siRNA-Cyr61 transfection. Transmigrated cells were detached after 22 h and counted in a hemocytometer. The number of siRNA-Cyr61 transmigrated cells was expressed as percentage of siRNA-Control transmigrated cells (100%). D. Cultures of MDA-MB-231-Tet-On-shRNA-c-Src in absence of Doxy were treated with 4 μg/ml of anti-Cyr61 (Cyr61) or with the corresponding amount of normal rabbit serum (Ctrl) for 48 h. E. Cultures of SUM159PT were treated with 1 μg/ml of anti-Cyr61 or with the corresponding amount of normal rabbit serum (Ctrl) for the last 20 h. Analyses of wound-healing were made at 0 and 20 h as described in Materials and Methods. Results are expressed as mean percentage of wound healing area ± SD at 20 h respect to 0 h from three independent experiments performed in triplicate (*p < 0.05, **p < 0.01).