Figure S4.
The effect of Lpg2525 expression on early endosomal morphology and the kinetics of RabGAP-5 or translocated Lpg2525 on the LCV. (a) HeLa-FcγRII cells were transfected with a plasmid-encoding GFP, GFP-Lpg2525 (full), or GFP-Lpg2525 (∆FB). At 24 h after transfection, cells were fixed and stained with anti-EEA1 antibody and DAPI. Asterisks indicate cells expressing GFP or the GFP-fusion proteins. Bar, 5 µm. Data are representative of three independent experiments (50 cells expressing the GFP constructs were scored in each experiment). Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (b) HeLa-FcγRII cells expressing GFP-RabGAP-5 were infected with the indicated L. pneumophila Lp01 strains for the indicated times at an MOI of 20. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria and then permeabilized and further stained with DAPI for detection of intracellular bacteria. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (c) HeLa-FcγRII cells were infected with the ∆lpg2525 L. pneumophila Lp01 strain complemented with 3x-FLAG- or 3x-FLAG-Lpg2525–expressing plasmids for the indicated times at an MOI of 20. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria and then permeabilized and further stained with DAPI and FLAG antibody for detection of intracellular bacteria and bacterially expressing FLAG-fusion proteins. Bar, 5 µm. (d) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting RabGAP-5. At 48 h after transfection, the cells were transfected with the additional plasmids encoding GFP and GFP-RabGAP-5 for 24 h. The efficacy of siRNA was assessed by IB with GFP antibody. IB with α-tubulin antibody was conducted as an internal control. IB, immunoblotting. Source data are available for this figure: SourceData FS4. Refer to the image caption for details.

The effect of Lpg2525 expression on early endosomal morphology and the kinetics of RabGAP-5 or translocated Lpg2525 on the LCV. (a) HeLa-FcγRII cells were transfected with a plasmid-encoding GFP, GFP-Lpg2525 (full), or GFP-Lpg2525 (∆FB). At 24 h after transfection, cells were fixed and stained with anti-EEA1 antibody and DAPI. Asterisks indicate cells expressing GFP or the GFP-fusion proteins. Bar, 5 µm. Data are representative of three independent experiments (50 cells expressing the GFP constructs were scored in each experiment). Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (b) HeLa-FcγRII cells expressing GFP-RabGAP-5 were infected with the indicated L. pneumophila Lp01 strains for the indicated times at an MOI of 20. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria and then permeabilized and further stained with DAPI for detection of intracellular bacteria. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (c) HeLa-FcγRII cells were infected with the ∆lpg2525 L. pneumophila Lp01 strain complemented with 3x-FLAG- or 3x-FLAG-Lpg2525–expressing plasmids for the indicated times at an MOI of 20. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria and then permeabilized and further stained with DAPI and FLAG antibody for detection of intracellular bacteria and bacterially expressing FLAG-fusion proteins. Bar, 5 µm. (d) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting RabGAP-5. At 48 h after transfection, the cells were transfected with the additional plasmids encoding GFP and GFP-RabGAP-5 for 24 h. The efficacy of siRNA was assessed by IB with GFP antibody. IB with α-tubulin antibody was conducted as an internal control. IB, immunoblotting. Source data are available for this figure: SourceData FS4.

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