Figure 6. NPRL2 enhances the effect of cisplatin that can activate cell cycle checkpoints.

H1229 cells treated with empty vector + IC₂₀ dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC₂₀ dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. (A) Cdc25A was degraded by the treatment of NPRL2 or IC₂₀ dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. P-Cdc2 and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. (B) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC₂₀ value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.