Effect of relaxin-2 knockdown with siRNA on invasion and angiogenesis. A, invasiveness of MG-63 cells transfected with relaxin-2 siRNA in Matrigel invasion assay. Cells were transfected with mock siRNA or relaxin-2 siRNA. After 48 h, 1 × 105 cells were allowed to invade through transwell inserts (8 Am) coated with Matrigel. The cells on lower surface of chambers were stained, counted, and photographed under a light microscope. The in vitro inhibition of invasiveness was calculated in five random 200-fold fields using the following formula: percent inhibition = (mock siRNA-relaxin-2 siRNA) /mock. Columns, mean from three separate experiments; bars, (*P < 0.01). B, HMEC endothelial cells treated with conditional medium from MG-63 cells after siRNA transfection by in vitro angiogenesis assay. The conditioned medium of MG-63 cells was collected after 48-h transfection following filtering of medium. HMEC-1endothelial cells seeded in eight-chamber slides were cultured with the above medium for 72 h until the formation of capillary network was observed. In the end of the experiment, angiogenesis was assessed by H&E staining and photographed under a microscope. columns, mean from three separate experiments; bars, SD (*P < 0.01).