Fig. 2.

Downregulating or upregulating the expression of RIPK4 enhanced or inhibited, respectively, the migratory and invasive abilities of BC cells in vitro and in vivo. a T24 and RT4 cells were infected with lentivirus-expressing RIPK4 shRNA-1, shRNA-2, shRNA-3 and shRNA-4, or a control shRNA; the RIPK4 protein level as measured by western blot. b Wound healing assays showing that RIPK4-silenced T24 and RT4 cells have lower motility compared to that of the control cells. c Matrigel invasion assays showing that RIPK4-silenced T24 and RT4 cells have decreased invasive capacity compared to that of the control cells. d Representative images of human umbilical vein endothelial cells (HUVECs) cultured with conditioned medium derived from RIPK4-silenced cells and control shNC cells. e Silencing of RIPK4 suppressed T24 and RT4 cell invasion and metastasis in severe combined immunodeficient (SCID-Beige) mice in vivo. Upper panel: Examples of haematoxylin and eosin staining in four lung nodule samples originating from T24-vector, T24-shRIPK4, RT4-vector, RT4-shRIPK4 cell-injected mice. Original magnification, ×200. Lower panel: Number of metastases in the lungs of mice (n = 7 per group) 8 weeks after tail injection of scrambled control shRNA T24 and RT4 cells (red; mean ± SEM, 13.3 ± 4.1 for T24, 14.4 ± 7.0 for RT4), and RIPK4 shRNA T24 and RT4 cells (blue; mean ± SEM, 6.2 ± 2.8 for T24, 3.4 ± 2.8 for RT4). The nodules were examined under an anatomical microscope. f Ectopic expression of RIPK4 was substantially increased in BIU87-RIPK4 cells compared with that in BIU87-vector cells by western blot. g Wound healing assays demonstrating that BIU87-RIPK4 cells had higher motility than BIU87-vector cells. h Ectopic overexpression of RIPK4 enhanced BIU87 cell invasion in a transwell assay. I Representative images of human umbilical vein endothelial cells (HUVECs) cultured with conditioned medium derived from RIPK4-overexpressing cells and vector control cells. j Overexpression of RIPK4 promoted BIU87 cell invasion and metastasis in severe combined immunodeficient (SCID-Beige) mice in vivo. Upper panel: Examples of haematoxylin and eosin staining in four lung nodule samples originating from BIU87-vector and BIU87-RIPK4 cell-injected mice. Original magnification, ×200. Lower panel: Number of metastases in lungs of mice (n = 7 per group) 8 weeks after tail injection of BIU87-vector cells (red; mean ± SEM, 3.0 ± 2.2) and BIU87-RIPK4 cells (blue; mean ± SEM, 11.1 ± 4.9). The nodules were examined under an anatomical microscope. *P < 0.05, **P < 0.01 by Student’s t test