FIG 2.

Inhibition of NOX2 and ROS enhances acidification of the lysosome-fused GAS-containing compartment, which is surrounded by LC3. (A and B) After prestaining with LysoTracker (200 nM) in the presence or absence of DPI (10 μM) or NAC (10 mM) for 1 h, the HMEC-1 cells were infected with GAS at an MOI of 5 for 30 min, and then gentamicin was added to kill extracellular bacteria. Cells were fixed at 3 h postinfection and stained with anti-LC3 and anti-LAMP-1 antibodies. DAPI was used for cell nuclear and bacterial DNA staining. Specimens were examined by confocal microscopy (A). LysoTracker (LysoT) stained strongly in the LC3- and LAMP-1-positive compartment after treatment with DPI (10 μM) or NAC (10 mM) in HMEC-1 cells (areas surrounded by a dashed line). Data represent the means ± SD from three independent experiments, and more than 100 cells were counted in each sample (B). Scale bar, 10 μm. (C) HMEC-1 cells with or without 1-h pretreatment with bafilomycin A1 (Baf A1) (100 nM) and/or DPI (10 μM) were infected with GAS at an MOI of 5 for 30 min and then treated with gentamicin to kill extracellular bacteria. Cells were collected at 1 and 6 h postinfection. The colony forming assay was performed to quantify the numbers of bacteria, and the fold values of GAS replication were calculated by normalizing the GAS count at 6 h with that at 1 h postinfection. Data represent the means ± SD from three independent experiments. **, P < 0.01; ***, P < 0.001.