PAO-induced ROS generation is involved in DNA damage and autophagic responses in HEK 293 cells. A: ROS generation was analyzed using CM-H2DCFDA fluorescent probe in PAO-treated HEK 293 cells with or without N-acetyl-l-cysteine (NAC, 10 mM). Bars = 25 µm. B: immunofluorescence microscopy showing dose-dependent enhancement of nuclear p-H2A.X in PAO-treated HEK 293 cells. Bars = 25 µm. C: Western blot analysis of beclin-1, ATG7, ATG5, and LC-3A/B-II in vehicle- vs. PAO-treated HEK 293 cells. D: Western blot analysis of p-CHK-1, p-H2A.X, and LC-3A/B-II in PAO-treated HEK 293 cells with or without NAC. β-Actin was used as a loading control. E: coimmunofluorescence staining for p-H2A.X and TUNEL-positive HEK 293 cells. Bars = 25 µm. Photomicrographs were captured using an Olympus BX51 microscope with an Olympus DP71 digital camera.