USP47-mediated deubiquitination and stabilization of YAP contributes to the progression of colorectal cancer

摘要

. (A) YAP interacts with USP47. Flag-YAP and Xpress-USP47 expression plasmids were co-transfected into HEK293T cells, the interaction between YAP and USP47 was determined by immunoprecipitation with α-Flag beads (top) or α-Xpress beads (bottom) followed by immunoblotting with α-Xpress or α-Flag antibody. One percent of whole cell lysates were loaded as input control. (B) USP47 deubiquitinates YAP in cells. Xpress-USP47, Flag-YAP and HA-Ub expression plasmids were co-transfected into HEK293T cells. The ubiquitination of precipitated YAP was analyzed by immunoblotting with anti-HA antibody. (C) USP47 deubiquitinates endogenous YAP. Xpress-USP47 expression plasmids were transfected into HEK293T cells, the ubiquitination of precipitated endogenous YAP was analyzed by immunoblotting with anti-ubiquitin antibody. (D) Knockdown of USP47 promotes YAP ubiquitination. Flag-YAP and HA-Ub expression plasmids were co-transfected into HEK293T-shCtr or HEK293T-shUSP47 cells, and cells were treated with MG132 (20 µmol/L). The ubiquitination of precipitated YAP was analyzed by immunoblotting with anti-HA antibody. (E) Knockdown of USP47 promotes endogenous YAP ubiquitination. Endogenous YAP was immunoprecipitated fromHEK293T-shCtr or HEK293T-shUSP47 cells pretreated with MG132 (20 µmol/L). The ubiquitination of YAP was analyzed by immunoblotting with anti-ubiquitin antibody. (F) Knockdown of USP47 promotes YAP degradation. HEK293T-shUSP47, HCT116-shUSP47 and HT29-shUSP47 cell lines and control cells were treated with or without MG132 (20 µmol/L). The expression levels of USP47, YAP and actin were determined. (G) mRNA expression analysis of YAP target genes from GEO dataset GDS2609 of colon mucosae from early onset CRC patients and healthy controls. (H and I) Knockdown of USP47 dramatically decreased YAP protein level and its target genes expression. The protein expression levels of USP47, YAP and actin were determined with antibodies indicated (H). The relative mRNA levels of USP47, YAP and YAP target genes were quantified using RT-qPCR, = 3 (I)