FIGURE 7.

FNBP1L is not required for the maturation of the Salmonella-containing vacuole, and FNBP1L truncations act as dominant negatives. A, FNBP1L knockdown does not affect the maturation of the SCV. HeLa cells were transfected with control or FNBP1L siRNAs and infected with S. Typhimurium 48 h later. One hour postinfection the cells were fixed and stained for LAMP1. The percentage of LAMP1-positive SCVs observed for each siRNA is shown. No significant differences in LAMP1 accumulation, and hence SCV maturation, were observed. Data are shown as the means of two independent experiments ± SEM, counting at least 100 cells for each condition with p values calculated by Student's t test, compared to siControl values. The accompanying micrographs show representative high-magnification crops of LAMP1 (green in merge) and DNA to mark bacteria (blue in merge). B, FNBP1L knockdown does not affect the ubiquitination (Ubq) of internalized Salmonella. HeLa cells treated with control or anti-FNBP1L siRNAs were infected with S. Typhimurium as above; following fixation, cells were stained with anti-ubiquitin Abs and examined for bacterial ubiquitination. The number of bacteria colocalizing with ubiquitin staining were quantified and no significant differences were observed between control and FNBP1L-deficient cells. Data are shown as the means of two independent experiments ± SEM, counting at least 100 cells for each condition with p values calculated by Student's t test, compared to siControl values. The accompanying micrographs show representative high magnification crops of staining for ubiquitinated proteins (green in merge) and DNA to mark bacteria (blue in merge). C, FNBP1L truncations act as dominant negatives, reducing autophagic efficiency against S. Typhimurium. LC3-GFP HeLa cells were transfected with 500 ng of Myc vector, full-length FNBP1L, or one of two truncations corresponding to amino acids 1–293 or 110–547 of the full-length protein. Twenty-four hours later the cells were infected with S. Typhimurium and fixed after 1 h. The number of bacteria captured within autophagic vacuoles was quantified as previously detailed. Significant loss of autophagy was observed with both truncated constructs when compared with full-length FNBP1L expression. Data are shown as means from at least 120 cells per condition ± SEM, with p values calculated by Student's t test, compared to full-length FNBP1L values.