Fig. 6

MAST3 is involved in the YAP-degradation process mediated by ubiquitin-proteasome pathway. (A). GSEA analysis showed that MAST3 is involved in ubiquitin-mediated protein degradation process. (B, C). siRNA-MAST3 and MAST3 plasmids were transfected into MCF-7 and MDA-MB-468, respectively. After 36 h, cycloheximide (CHX, 532.5 nM) was added and proteins at different time points were collected for western blot analysis. GAPDH served as an internal reference protein. (D, E). MAST3 mediated the metabolic curve of YAP protein degradation. *: P < 0.05. (F). MDA-MB-468 cells were transfected with GFP-tagged YAP plasmid, 24 h later, the cells were added to DMSO or MG132 (concentration: 20 µM) for different time points (including 4 and 8 h, respectively). Cells were lysed and subjected to western blot. GAPDH serves as a loading control. (G): GFP-YAP plasmid and Myc-MAST3 plasmid were co-transfected into MDA-MB-468, and after 24 h, DMSO or MG132 (20 µM) was added. After 24 h of treatment, the protein collected was subjected to western blot analysis. GAPDH served as an internal reference protein. (H and I). MAST3 wild type-plasmid or its mutant plasmids (delta-kinase domain and delta-PDZ domain), along with HA-ubiquitin (Ub) plasmid were co-transfected in MDA-MB-468 cells for 48 h (H), shRNA-MAST3 and HA-Ub plasmid were co-transfected in MCF-7 cells for 72 h, respectively (I). Cells were lysed and the changes in YAP ubiquitination level was subjected to immunoprecipitation and western blot. (J). The association between MAST3 and YAP expression in multiple breast cancer cell lines was detected using western blot. GAPDH served as an internal reference protein. (K). Representative photographs of the correlation between MAST3 and YAP expression in human breast cancer specimens (magnification, 200×, scale bar: 100 μm; magnification, 400×, scale bar: 50 μm). Paired Student t-test, Columns: mean numbers, Bars: SD, each experiment was performed triplicate