FIG 5 .

Both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination, and Gal-8 knockdown reduces recruitment of parkin to GAS. (A and B) Gal-8 (A) and parkin (B) expression was silenced in A549 cells using three lentivirus-based shRNAs (shGal8 clones 1, 2, and 3 and shParkin clones 1, 2, and 3). Luciferase shRNA (shLuc) was used as a negative control. The expression of Gal-8 and parkin was detected by Western blot analysis. (C) shLuc-A549, shGal8-A549 (clone 2), and shParkin-A549 (clone 2) cells were infected with GAS at MOI = 25 for 30 min, and gentamicin was added to kill extracellular bacteria. Cells were collected at 1 h postinfection and stained with anti-Gal-8, anti-parkin, and anti-ubiquitin (UB) antibodies. DAPI was used for cell nuclear and bacterial DNA staining. Images were observed by confocal microscopy. Scale bar, 10 μm. (D and E) Levels of GAS surrounded with UB (D) and parkin (E) were determined relative to total levels of intracellular GAS. All quantitative data represent the means ± SD of results from three independent experiments, and over 100 cells were counted in each sample. **, P < 0.01, ***, P < 0.001 (compared to shLuc cells). (F) shLuc-A549, shGal8-A549, and shParkin-A549 cells were further infected with GAS at MOI = 25 for 30 min, and gentamicin was added to kill extracellular bacteria. Cells were collected at 1 and 6 h postinfection. Bacteria were quantified by colony-forming assay, 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 of results from three independent experiments. *, P < 0.05, ***, P < 0.001 (compared to shLuc cells).