Fig 4. p47^phox mediates IFN-γ-induced ROS generation and a mimic ETosis.

(A) A representative Western blot of the indicated proteins from A549 cells transfected with shRNA targeting luciferase (shLuc) and shRNA targeting p47^phox (shp47^phox) (clones 1 to 5). β-actin was used as an internal control. The relative ratios of p47^phox and β-actin are also shown as the mean ± SD of three independent experiments. ***P < 0.001. (B) ROS generation in IFN-γ (10 ng/ml)-treated shLuc- and shp47^phox (clones 1 and 2)-transfected A549 cells for 6 days was determined using CM-H₂DCFDA staining followed by analysis using a fluorescent plate reader. The data are presented as the mean ± SD of triplicate cultures and are shown as relative optical densities (O.D.). ***P < 0.001. Fluorescence microscopy was used to obtain representative images of DAPI-stained nuclei and to determine the percentage of cells with mimic ETosis (C). Nucleosome detection was measured using ELISA (D). The data for the percentages of cells with mimic ETosis and the O.D. value of nucleosome detection are shown as the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001. (E) Cell viability was assessed using the trypan blue exclusion test. The data are presented as the mean ± SD of triplicate cultures and are shown as relative percentages. **P < 0.01. (F) shRNA targeting gp91^phox was used to silence gp91^phox as detected by Western blot analysis. The expression of nucleosome and the percentages of cells with mimic ETosis in gp91^phox-transfected A549 cells with or without IFN-γ (10 ng/ml) treatment were determined accordingly. The data for the O.D. value of nucleosome detection and the percentages of cells with mimic ETosis are shown as the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001.