Fig. 5. Inhibition of iPLA₂ induces accumulation of p53 and expression of p21^cip1. (A) Expression of ARD-iPLA₂-GFP in INS-1 cells induces the accumulation of p53 and expression of p21. Cells were mock-transfected or transfected with increasing amounts of DNA (2, 5 and 10 µg/100 mm plate). Cell lysates were prepared and analyzed by western blotting with antibodies against GFP, p53, p21 and actin. Recombinant TNT p53 and ARD-iPLA₂-GFP proteins were loaded as controls. (B) Treatment with BEL for 30 hours increases p53, p21 and p27 levels and decreases cyclin A levels in INS-1 cells. Cells were treated with or without BEL (15 µM) for 30 hours. Cell lysates were prepared and analyzed by western blotting for p53, p21, p27, cyclin A and actin. (C) p53 accumulates in the nuclei of BEL-treated INS-1 cells. Cells treated with 15 µM BEL were fixed and stained with anti-p53 (FL-393, Santa Cruz Biotechnology) and counterstained with DAPI followed by analysis with a confocal scanning microscope (Zeiss LSM 510 META). Magnification, 100x.