FIG 3.

Inhibition of NOX2 and ROS switches LAP to autophagy during GAS infection. (A to E) In the presence or absence of DPI (10 μM) or NAC (10 mM) for 1 h, HMEC-1 cells were infected with GAS at an MOI of 5 for 30 min, and then gentamicin was added to kill extracellular bacteria. (A and B) Cells were fixed at 1 h postinfection and stained with anti-ULK1 and anti-LC3 antibodies. DAPI was used for cell nuclear and bacterial DNA staining. Specimens were observed by confocal microscopy (A). Scale bar, 10 μm. LC3-positive GAS surrounded with ULK1 was relative to total intracellular LC3-positive GAS (B). Data represent the means ± SD from three independent experiments, and over 100 cells were counted in each sample. **, P < 0.01; ***, P < 0.001. (C) Cells were collected at 1 h postinfection, followed by Western blot analysis to determine the protein expression levels of phosphorylated p70s6k Thr389 and total p70s6k. (D) Cells were fixed at 1 h postinfection, and conventional TEM was performed to observe the membrane structure of GAS-containing vacuoles. White arrowheads indicate the single-membrane structure; black arrowheads indicate the double-membrane structure. IM, isolation membrane. (E) Cells were collected at 1 h postinfection, followed by Western blot analysis to determine the protein expression levels of phosphorylated AKT Ser473, total AKT, phosphorylated ERK1/2 Thr202/Tyr204, and total ERK. (F) After pretreatment or no pretreatment with LY294002 (25 μM), AKT inhibitor X (25 μM), and PD98059 (25 μM) for 1 h, cells were infected with GAS at an MOI of 5 for 30 min, and then gentamicin was added to kill extracellular bacteria. Cells were collected at 3 h postinfection, followed by Western blot analysis to determine protein expression levels of phosphorylated p70s6k Thr389 and total p70s6k. β-Actin was used as an internal control for Western blot analysis.