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. 2015 Nov 13;7(1):638–655. doi: 10.18632/oncotarget.6313

Figure 7. EMQA reactivates PP2A in NSCLC cells by disrupting the SET-PP2Ac binding.

Figure 7

(A) Does-dependent effects of EMQA on SET-PP2Ac binding. After exposure to EMQA treatment at the indicated doses, A549 cells were harvested and analyzed by co-immunoprecipitation. The expression of PP2Ac in the SET-immunoprecipitant complex (middle panel) and the expression of SET in the PP2Ac-immunoprecipitant complex (right panel) decreased with increasing dose of EMQA. (B) Time-dependent effects of EMQA on SET-PP2Ac binding. A549 cells were collected after exposure to 5 μM EMQA at the indicated times and analyzed by co-immunoprecipitation. Western blot analysis of the SET-immunoprecipitant complex showed that EMQA disrupted SET-PP2Ac binding in a time-dependent manner. (n = 3) (C) Proximal ligation assay (PLA) revealed that EMQA plus paclitaxel treatment significantly reduced the SET-PP2Ac binding in A549 cells. (Red dots in PLA image, nucleus stained with DAPI in blue) The corresponding quantification is shown on the right side. Bar: mean, error bar: S.D. (D) Ectopic expression of SET-myc diminished the effects of EMQA and paclitaxel combination treatment on inhibition of p-Akt and apoptosis induction. After transfecting A549 and H460 cells with SET-myc for 24 hours, cells were treated with EMQA 5 μM and paclitaxel 10 nM for 24 hours and analyzed by flow cytoemetry and western blot. Bar, mean; error bars, S.D. (n = 3) (E) The 227–277 sequence at the C-terminal of SET protein is critical for the effect of EMQA. Four different recombinant truncated SET proteins (as shown in Figure 7F, left panel), SET1–127, SET1–177, SET1–227 and SET76–277 were generated and mixed the full-length SET protein in the indicated proportions. Under a fixed concentration of EMQA, the binding affinities of the indicated protein mixture to PP2Ac were detected in a BIAcore T200 system. (n = 3) The symbolic binding relationships between SET proteins and PP2Ac were illustrated. (F) After transfecting A549 cells with vectors coding full-length SET-DDK, SET1–127-DDK, SET1–177-DDK, SET1–227-DDK and SET76–277-DDK or 24 hours, these cells were treated with EMQA 5 μM and analyzed by co-immunoprecipitation. Co-immunoprecipitation analysis showed that EMQA significantly decreased the SETFL-PP2Ac and SET76–277 binding, while the association of PP2Ac and other truncated SET proteins were not affected. Left panel showed the schema of the truncated SET proteins.