FIGURE 6.

FNBP1L localizes to a subset of internalized bacteria; ATG3 localization to Salmonella is FNBP1L dependent. A, FNBP1L-GFP-transfected cells and control GFP-expressing cells were infected with S. Typhimurium for 40 min, fixed, and examined by confocal microscopy. A subset of internalized S. Typhimurium was surrounded by FNBP1L-GFP “halos” (arrows); these structures were absent in GFP-expressing cells. In merged images S. Typhimurium is shown in blue and FNBP1L-GFP/GFP is green; the scale bar represents 10 μm. Images were cropped and were taken from high-resolution confocal z-projections. B, Colocalization of FNBP1L and S. Typhimurium was observed to peak at 40 min postinfection, with a marked drop at 1 h. The peak of 21% colocalization is of a similar magnitude to that of S. Typhimurium and LC3-GFP at 1 h. Data are shown as means and SEM and were pooled from two independent experiments, each counting at least 50 cells per condition. C, Localization of ATG3-GFP to S. Typhimurium is FNBP1L dependent. HeLa cells were cotransfected with control or FNBP1L-directed siRNA and ATG3-GFP plasmid and infected 48 h later. Forty minutes postinfection the cells were fixed, stained, and examined for ATG3-GFP/bacterial association. In control cells, 10% of internalized S. Typhimurium were placed within ATG3-GFP “halos,” and this percentage dropped to 1% in the absence of FNBP1L. Data are presented as means with SEM; data were pooled from two independent experiments, each counting at least 50 cells per condition. Significance was assessed using one-way ANOVA, followed by post hoc t tests using the Bonferroni correction, comparing control siRNA to FNBP1L-directed siRNA samples. D, ATG3-GFP and FNBP1L-Myc colocate to a subset of bacteria within infected cells. HeLa cells cotransfected with ATG3-GFP and FNBP1L-Myc were infected with S. Typhimurium for 40 min and fixed and stained. Upper row shows overview of a confocal z-stack projection with the lower row showing high magnification crops of the area marked in the merge column. ATG3-GFP is shown as green, FNBP1L-Myc as red, and DNA as blue in the merged images. Bacteria surrounded by both FNBP1L and ATG3 are marked by arrows in the lower panelS. Scale bar represents 10 μm.