FIGURE 5. ROS-regulated SHP2 inhibited Pa-activated STAT1 in the absence of Atg7.

(A) MH-S cells were infected with PAO1 (MOI=10) for different times. H₂O₂ production was determined using EuTc assay (absorbance at 617 nm). (B) MH-S cells were transfected with Ctrl siRNA or Atg7 siRNA for 24 h, then infected with PAO1 (MOI=10, 2 h). H₂O₂ was measured as above. (C) MH-S cells were pretreated with DPI (5 μM) for 30 min. EuTc assay was used to measure H₂O₂ after INF-γ or Pa infection. (D) MH-S cells were pretreated with H₂O₂ (10 mM) or DPI (5 μM) for 30 min. Griess reagent was used to detect the generation of nitrite after IFN-γ or Pa infection. (E) Immunoblotting was used to determine NOS2 and phosphorylation of STAT1α after Pa infection. (F, G) 24 h after transfected with Ctrl siRNA or SHP2 siRNA, or double knocked down using Atg7 siRNA and SHP2 siRNA, MH-S cells were infected with Pa as above. Immunoblotting shows the expression of NOS2 and phosphorylation of STAT1α. (H) Bacterial killing of Pa by MH-S cells treated with H₂O₂ either with Ctrl siRNA, SHP2 siRNA or Atg7 siRNA transfection. (I) Cell viability was measured by MTT assay. Data are representative as means±SD of three independent experiments (*, p<0.05; **, p<0.01).