FIG 3 .

Intracellular persistence of S. aureus USA300 in keratinocytes treated with inhibitors of caspases and endosomal acidification. (A and B) Intracellular persistence of WTS. aureus USA300 (A) and USA300 agr null mutant (B) in the presence of caspase-1 or caspase-3 inhibitors in HaCaT and HEKn cells. (C) Effects of chloroquine and bafilomycin pretreatment on intracellular persistence. Values in panels A to C are the means plus standard deviations (SD) (error bars) of sextup licate wells. (D and E) Light micrographs (D) and electron micrographs (E) of WT and agr mutant-infected organotypic cultures (skin grafts maintained on fibroblast feeder cells), showing accumulation of staphylococci in the stratum granulosum (black arrows) and organisms that do not appear to be within a membrane-bound compartment. The photomicrographs in panels D and E are representative images of infections using at least three separate grafts and multiple sections of each graft. (F) Confocal images of infected HaCaT cells with fluorescent labeling of EEA-1 (early endosome [green]) and MDC (autophagosome [green]); demonstrating some colocalization (yellow) of both WT S. aureus and S. aureus agr mutant (red) with the autophagosomal MDC marker, but not with the early endosome EEA-1 at 24 h postinfection. Intracellular persistence assays were performed at least three times, and the results of a representative experiment are shown. Confocal imaging was done at least twice on separate samples, and representative images are shown.