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Results: 4

1.
Figure 4.

Figure 4. From: The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila.

fob is required for the fusion of phagosomes with lysosomes. Micrographs show hemocytes isolated from wild-type (A–E) or fob1 (A’–E’) larvae. (A–C) Hemocytes were allowed to phagocytose FITC-labeled E. coli, and were immunostained for Avl (red) and Rbsn-5 (blue; A), Rab7 (B), or Hook (C). (D) Hemocytes were allowed to internalize dextran–Alexa Fluor 488 (10 kD), which after a 90-min chase partially colocalized with LysoTracker in wild type (D) and fob1 (D), indicating functional labeling of lysosomes by dextran. (E) Hemocytes with lysosomes preloaded with internalized Texas red–dextran were allowed to phagocytose GFP-labeled E. coli. After 30–45 min of chase, the bacteria colocalized with dextran in wild-type (E) but not fob1 hemocytes (E′). For display, images were imported into Photoshop (Adobe) and adjusted for gain, contrast, and gamma settings. Bar, 5 µm. (F) Bar graphs indicate percentages of bacteria in phagosomes positive for the indicated markers or the percentage of dextran in LysoTracker-positive structures (error bars indicate standard deviation). Hemocytes were harvested from larvae with indicated genotypes.

Mohammed Ali Akbar, et al. J Cell Biol. 2011 February 7;192(3):383-390.
2.
Figure 3.

Figure 3. From: The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila.

fob phagosomes fail to mature. Double-labeled bacteria were allowed to internalize for 10 min (broken line) or 30 min (solid line), and images were captured for 15 min. The distribution of fluorescence ratios is shown for Ore-R (A) and fob1 (B). The fluorescence ratio relates to pH as shown in Fig. S1. (C) Electron micrographs of phagosomes detected after a 30-min chase of phagocytosed E. coli. Phagosomal structures were broadly classified in three categories based on their ultrastructural appearance: phagosome (C), late phagosome (C′) and phagolysosomes (C′′). Bar, 1 µm. (D) Relative distribution of different categories of phagosomes in Ore-R and fob1. Data were collected from two independent sets of experiments with equivalent results. Quantification was performed in triplicate with a representative example shown in D.

Mohammed Ali Akbar, et al. J Cell Biol. 2011 February 7;192(3):383-390.
3.
Figure 2.

Figure 2. From: The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila.

fob mutants exhibit a defect in bacterial clearance. (A) 2 h after injection, pHrodo-labeled E. coli (arrows) were visible around the dorsal vessel in the thorax of wild-type and eater flies (Df(3R)D605/Df(3R)Tl-I). In contrast, diffuse weaker signals appeared in fob1 flies. (B) Hemocytes were allowed to engulf FITC-labeled E. coli for 15 min, and, after quenching fluorescence of external bacteria with Trypan blue, the fluorescence of phagocytosed bacteria was visible in wild-type (B) and fob1 (C) cells visualized by differential interference contrast microscopy (B′ and C′). (D and E) After 45 min of chase, bacteria were cleared from wild type (D) but accumulated inside fob1 hemocytes (E). (F) Box and whisker plots display the number of bacteria detected in hemocytes of indicated genotypes after 45 min of chase. Bars: (A) 0.5 mm; (B and C) 25 µm; (D and E) 10 µm.

Mohammed Ali Akbar, et al. J Cell Biol. 2011 February 7;192(3):383-390.
4.
Figure 1.

Figure 1. From: The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila.

A fob null allele is hypersensitive to infections with E. coli. (A) Schematic representation of the Drosophila fob gene and its neighbors. The targeting fragment generated in vivo (Gong and Golic, 2003) contains portions of neighboring genes around the mini-white gene (red box). (B) Southern hybridization with the entire fob gene yielded a signal with Ore-R (wt) but with any fob allele. Reprobing the same membrane with dVps33b confirmed the presence of DNA in all lanes. (C) qRT-PCR showed no expression of fob but similar levels of expression for neighboring genes in fob1 compared with wild type. Gene expression levels were normalized with rp49 as an internal control and are shown relative to wild type. (D and E) Survival after infection was measured for wt, fob1, and rescued fob1 flies after injection with E. coli (D) or E. faecalis (E). (F) Induction of AMP genes 6 h after injection with E. coli (Drosocin, Diptericin, Cecropin, and Attacin) or 12 h after injection with E. faecalis (Defensin). (G) Bacterial load in injected flies at the indicated day after injection with E. coli. Error bars indicate standard deviation.

Mohammed Ali Akbar, et al. J Cell Biol. 2011 February 7;192(3):383-390.

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