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Results: 5

1.
Fig. 1.

Fig. 1. From: Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.

T3SSBsa is required for endosome escape but not invasion. (A) Plaque-forming units (pfu) per bacteria 18 h after infection of HEK293 cells with BtE264, Bp340, ΔsctN mutants, or complemented derivatives (psctN). Dashed line, limit of detection. (B) Colony forming units (cfu) recovered per bacteria in 2-h invasion assays. HEK293 cells were untreated (solid bars) or treated with 2 μg/mL cytochalasin D (shaded bars) before infection. Assays were performed in triplicate and error bars represent ±SEM. *P < 0.005.

Christopher T. French, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):12095-12100.
2.
Fig. 4.

Fig. 4. From: Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.

T6SS-1 is critical for efficient intercellular spread. (A) Invasion efficiencies by BtE264 or ΔclpV1 mutants 2 h postinfection in HEK293 cells. (B) HEK293 cells were infected and stained for bacteria (red) and actin (green) 8 h postinfection. (Scale bar, 20 μm.) (C) Time course of intracellular replication in HEK293 cells. (D) Plaque-forming efficiency on HEK293 cell monolayers 18 h after infection. (E) Cytotoxicity assays in HEK293 cells. All assays in A and CE were performed in triplicate and error bars represent ±SEM. **P < 0.05; *P < 0.005.

Christopher T. French, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):12095-12100.
3.
Fig. 2.

Fig. 2. From: Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.

Intercellular spread following photothermal nanoblade-mediated delivery. (A) Upper, invasion by BtE264 is followed by T3SSBsa-mediated endosome escape and BimA-mediated actin polymerization in the cytoplasm. Lower, photothermal excitation of Ti-coated microcapillary pipettes using a 6-ns, 532-nm laser pulse facilitates pressurized delivery of bacteria directly into the cytosol. (B) Plaque formation on HEK293 monolayers after infection with BtE264 (Top), ΔsctN (Middle), or ΔbimA mutants (Bottom). (Scale bar, 1 cm.) (C) Bt and mutants (red) were delivered into HEK293 cells using a photothermal nanoblade and stained for actin (green) 12 h later. (Scale bar, 20 μm.) (D) Plaque formation on HEK293 monolayers following nanoblade delivery. (Scale bar, 1 cm.) (E) Plaques in D stained for bacteria (red) and actin (green). (Scale bar, 500 μm.) (F) Magnified edges of plaques in E. (Scale bar, 20 μm.)

Christopher T. French, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):12095-12100.
4.
Fig. 3.

Fig. 3. From: Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.

fla2-mediated flagellar motility facilitates plaque formation. (A) Fraction of HEK293 cells containing rapidly motile bacteria 8 h after infection with BtE264 and derivatives. Approximately 300 cells were monitored per strain. (B) Plaque-forming efficiency on HEK293 cell monolayers 18 h after infection. (C) Representative infected cells stained for bacteria (red) and actin (green). (D) Plaque diameters 24 h after infection of HEK293 cell monolayers. (E) HEK293 cell invasion efficiencies by BtE264 or mutant strains 2 h postinfection. (F) Time course of intracellular replication in HEK293 cells. All assays were performed in triplicate and error bars represent ±SEM. *P < 0.005; Un, undetectable.

Christopher T. French, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):12095-12100.
5.
Fig. 5.

Fig. 5. From: Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.

Intercellular spread and plaque formation occur through cell fusion. (A) Progression of events leading to plaque formation. (Left) HEK293-RFP (red) and -GFP (green) cells immediately after infection. Twelve hours later, a MNGC is formed (yellow, Center), which undergoes lysis and forms a plaque at 24 h (Right). (Scale bar, 500 μm.) (B) A MNGC (Left) was stained with DAPI (blue, Center). (Right) Triple-color merged image. (C) MNGC and plaque formation by BtE264 and mutants on HEK293 RFP + GFP monolayers 12 h (Upper) or 24 h (Lower) following infection or nanoblade delivery (“n”, ΔsctN, ΔsctNΔclpV1). (D) MNGC and plaque formation 12 h (Upper) and 24 h (Lower) after infection with Bp340 and mutants. (E) Plaque formation 56 h following infection with L. monocytogenes 10403S. The slight appearance of yellow at the edge of plaques is due to physical overlap of red and green cells and does not indicate cell fusion. Images are representative of multiple independent experiments.

Christopher T. French, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):12095-12100.

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