Fig. 4

RBCK1 inhibits YAP protein level and Hippo target gene expression in TNBC A–B RBCK1 depletion increases the level of YAP protein in TNBC cells. MDA-MB-231 and BT549 cells were transfected with siControl or siRBCK1. 48 h later, the cells were collected for western blot analysis. C–D RBCK1 deletion increases expression of YAP target gene in TNBC cells. MDA-MB-231 and BT549 cells were transfected with siControl or siRBCK1. 48 h later, total RNA was collected for gene expression analysis. 36B4 was used as internal control. Each group was analyzed three times. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. E–F RBCK1 deletion increased the activity of TEAD luciferase in TNBC cells. MDA-MB-231 and BT549 cells were transfected with siControl, siRBCK1,YAP luciferase reporter plasmid and Renilla plasmid. After 24 h, the cells were harvested 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 Overexpression of RBCK1 decreased the levels of YAP protein in HEK293 cells. HEK293 cells were transfected with Flag-RBCK1 or Flag-tag plasmids. 48 h later, the cells were collected for western blot analysis. H Overexpression of RBCK1 reduced the expression of YAP target gene in HEK293cells. HEK293 cells were transfected with Flag-RBCK1 or Flag-tag plasmids. After 48 h, total RNA was extracted for the gene expression analysis. 36B4 was used as internal control. Each group was analyzed three times. I–J Overexpression of RBCK1 reduced the expression of YAP target gene. MDA-MB-231 and BT549 cells with stable expression of RBCK1 or empty vector, total RNA was extracted for the gene expression analysis. 36B4 was used as internal control. Each group was analyzed three times. K–L Overexpression of RBCK1 reduced TEAD Luciferase activity in TNBC cells. MDA-MB-231 and BT549 cells with stable expression of RBCK1 or empty vector. After 24 h, the cells were transfected with the YAP luciferase reporter plasmid and Renilla plasmid. After 24 h, cells were harvested 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