Figure 9.

NOX4 gene knockdown suppressed AgNP-induced mitochondrial apoptosis and ROS generation in ARPE-19 cells. ARPE-19 cells were transfected with control siRNA or specific NOX4 siRNAs and treated with 5 μg/mL AgNPs for 24 h. (A) Cells were stained with NOX4 (green) antibody or CellROX deep red and detected under a confocal microscope. (B) Protein levels of NOX4 and PARP assessed by Western blot analysis. α-Tubulin was used as an internal loading control. (C) Mitochondrial ROS levels were measured by flow cytometry after MitoSOX staining. Data are expressed as the mean ± standard deviation of three experiments. **P < 0.01, ***, P < 0.001. (D) Cleaved caspase-3 was detected by confocal microscopy. (E) JC-1 stained cells were observed by confocal imaging. In JC-1 stained cells, red fluorescence is visible in cells with high mitochondrial membrane potential, whereas green fluorescence of JC-1 monomer is present in cells with low mitochondrial potential.

Abbreviations: AgNPs, silver nanoparticles; NOX4, NADPH oxidase 4; siRNA, small interfering RNA; PARP, poly (ADP‐ribose) polymerase; ROS, reactive oxygen species; JC-1, 5.5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; DAPI, 4ʹ,6-diamidino-2-phenylindole