Fig. 4

MAST3 is identified as a positive regulator of the Hippo pathway. (A–B): MAST3 overexpression vector and shRNA-MAST3 are transfected into MDA-MB-468 and MCF-7, respectively. Fluorescence reporter gene detection reveals that MAST3 significantly reduces the transcriptional activity of YAP-TEAD, with YAP acting as a signal amplifier. (C): Western blot analysis revealed that MAST3 significantly downregulates the amount of YAP protein, while the phosphorylation levels and content of upstream kinases MST-LATS did not show significant changes. GAPDH served as an internal reference. (D–E): RT-qPCR detection showed that MAST3 downregulates the mRNA levels of target genes CTGF, CYR61, and CCNE1 in the Hippo pathway but induced no significant change in the mRNA levels of YAP. (F): Immunofluorescence experiments showed that MAST3 overexpression in MDA-MB-468 weakened the ability of YAP to enter the nucleus, whereas MAST3 knockdown led to the opposite result. The areas (200×, scale bar: 50 μm) using red rectangle box labels were enlarged at the right side (400×, scale bar: 20 μm). Paired Student t-test, Columns: mean numbers, Bars: SD, each experiment was performed triplicate. *: P < 0.05; * *: P < 0.01; * * *: P < 0.001