Nutlin-3 induces apoptosis in ARPE-19 and primary RPE cells. (A) ARPE-19 cells were grown to confluence for 3 days, serum starved for 24 hours, and treated with 20, 40, and 60 μM Nutlin-3 for 2 hours. DNA fragmentation was measured by ELISA (mean ± SEM). *P < 0.05; significantly different compared with DMSO. (B) Western blot for active caspase-9 and -3 in 0, 20, 40, and 60 μM Nutlin-3–treated lysates. Actin was used as an internal loading control. (C) ARPE-19 cells were treated with DMSO or Nutlin-3 (60 μM) for 2 and 4 hours, and monolayers were photographed under a phase-contrast microscope equipped with a charge-coupled device camera using Image J software. (D) Primary RPE cultures were grown to confluence, serum starved for 24 hours, and treated with 0, 20, 40, and 60 μM Nutlin-3 for 2 hours. Lysates were analyzed by Western blot for the presence of active caspase-9 and -3. Actin was used as an internal loading control.