Fig. 5

RBCK1 inhibits TNBC cancer progression via Hippo/YAP axis A–B RBCK1 deletion boosted the level of YAP protein, and the function can be saved after YAP deletion in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP. 48 h later,the cells were collected for western blot analysis. C–D RBCK1 deletion increased the expression of Hippo target gene, which can be reversed after YAP knocking-down in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP. 48 h later, total RNA was collected for gene expression analysis. 36B4 was used as internal control. Each group was analyzed three times. E–F RBCK1 deletion increased the activity of TEAD luciferase, which can be reversed after YAP knocking-down in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP., YAP luciferase reporter plasmid and Renilla plasmid. 24 h later, the cells were collected for the detection of luciferase activity. Each group was analyzed three times. *P < 0.05; **P < 0.01; ***P < 0.001 for gene expression comparisons. G–H RBCK1 depletion promotes the migration of cell, which can be reversed after YAP knocking-down in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP. We used the trans-well assay analysis to examine the invasion capability of TNBC cell. Calculate the number of cells and the number data showed as ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 for cell comparisons. I–J RBCK1 depletion promotes the invasion of cell, which can be reversed after YAP knocking-down in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP. We used the trans-well assay analysis to examine the invasion capability of TNBC cell. Calculate the number of cells and the number data showed as ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 for cell comparisons. K–L Wound-healing experiment confirmed that RBCK1 depletion increased the migration capacity of TNBC cells, the function could be saved after YAP deletion in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP. Wound closure was quantified at a specified point in time. Data are showed as ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 for cell comparisons. M–N RBCK1 depletion inhibited apoptosis, which can be reversed after YAP knocking-down in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP. 24 h later, the cells were stained with PI and Annexin V. Then, FACS analysis was performed on the cells to determine the proportion of apoptotic cells. Each group was analyzed three times. O–P RBCK1 depletion increased the expression of CD24-CD44 + , in which can be reversed after YAP knocking-down in TNBC cell. MDA-MB-231 and BT459 cells were transfected with siControl, siRBCK1 or siRBCK1 + siYAP. After 24 h, cells were stained with anti-CD44-FITC and anti-CD24-PE. Then, FACS analysis was performed on the cells to determine the proportion of CD24-CD44 + cells. Each group was analyzed in three times