YDJC Induces Epithelial-Mesenchymal Transition via Escaping from Interaction with CDC16 through Ubiquitination of PP2A
Figure 6
YDJC downregulates PP2A expression, and CDC16 reverses it. (a) CDC16 induces and YDJC suppresses PP2A expression. The expression levels of PP2A and PP2A-C mRNA levels in the Control (siCon) or YDJC-knockdown (siYDJC) or CDC16-knockdown (siCDC16) or YDJC-overexpressing (YDJC) or CDC16-overexpressing (CDC16)-A549 cells were analyzed using western blotting or qRT–PCR. (b) Effects of siCDC16 on PP2A expression in YDJC stably silenced A549 cell lines. The expression levels of PP2A and PP2A-C mRNA levels in the Control (siCon) or CDC16-knockdown (siCDC16)-YDJC stably silenced A549 cells were analyzed using western blotting or qRT–PCR. Mean± SD; n=3. All P-values were determined based on two-tailed Student’s t test. In (a) and (b), A549 cells were transfected with the plasmid consisting of CDC16, YDJC, or an empty vector (4 μg), respectively. Gene silencing of CDC16 or YDJC was carried out by transfecting the A549 cells with the indicated amounts of siCDC16, siYDJC, or control siRNA. Cell lysates were prepared, and the protein level was resolved to 8-12% SDS-PAGE. The expression level of PP2A was determined by western blotting; β-actin was used as an internal control. qRT-PCR analysis of PP2A expression in cDNA derived from the indicated cells. GAPDH is included as a loading control. (c) Binding of CDC16 to PP2A and YDJC. Immunoprecipitation with the CDC16 antibody (IP: CDC16) was carried out with the cell lysates of A549 cells overexpressed with CDC16 plasmids. The resulting immunocomplexes were analyzed with Western blotting with PP2A, YDJC, or CDC16 antibodies, respectively. (d) Stimulatory effects of YDJC on ubiquitination of PP2A. Immunoprecipitation with the PP2A antibody (IP: PP2A) was done using cell lysates of A549 cells overexpressed with wild-type or plasmids. (e) Effects of CDC16 overexpression on PP2A ubiquitination. Immunoprecipitation with the PP2A antibody (IP: PP2A) was done using the cell lysates of A549 cells transfected with CDC16 plasmids. In d and e, the resulting immunocomplexes were detected by Western blotting with PP2A or ubiquitin (Ub) antibodies. (f) Effects of stably expressed CDC16 on YDJC ubiquitination in A549 cell lines. Immunoprecipitation with the YDJC antibody (IP: YDJC) was carried out using the cell lysates of A549 cells stably transfected with plasmids containing CDC16. The resulting immunocomplexes were detected by Western blotting with YDJC and Ub antibodies, respectively. (g) The proposed scheme of YDJC’s mechanism of action.