Figure 1.
SopB-mediated Akt activation at Salmonella invasion ruffles is PI(3,4) P2/PI(3–5) P3-dependent.
(A) HeLa cells were infected with wild type or ΔsopB mutant S. Typhimurium and fixed at 30 min p.i. Cells were examined by epifluorescence microscopy after co-staining for activated Akt with a phospho-specific (Ser473) antibody, actin with fluorescently-labeled phalloidin and bacteria with DAPI. Insets are enlarged from boxed areas. (B) HeLa cells were transiently transfected with GFP-PH-Akt for 16 h prior to Salmonella infection. Cells were infected and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. (C) HeLa cells were transiently transfected with GFP or PTEN-A4-YFP for 16 h prior to Salmonella infection. Cells were infected with wild type S. Typhimurium and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin, Akt with a phospho-specific (Ser473) antibody and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. Size bars, 10 µm. (D) The percentage of phospho-Akt+ Salmonella invasion ruffles from C was quantified (n ≥ 50). Averages ± SD for three separate experiments are shown. Asterisk indicates that the percent value is significantly different from the control (P < 0.001) as determined by one-way ANOVA analysis. (E) HeLa cells were transiently transfected with GFP-Akt and PTEN-A4-YFP or GFP for 16 h prior to infection. Cells were uninfected, infected as in A, or incubated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. GFP antibodies were used to ensure equal Akt transfection.
Figure 2.
SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor LY294002.
(A) HeLa cells were treated with different concentrations of LY294002 (1 µM to 100 µM) for 30 min. Cells were then infected with wild type S. Typhimurium for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with ΔsopB mutant S. Typhimurium for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type S. Typhimurium or EGF for three separate experiments, calculated as outlined in the Materials and Methods. Asterisks indicate that the percent value is significantly different from the control (P < 0.05).
Figure 3.
Class II PI3-Kinase beta isoform (PI3K-C2β) plays a role in SopB-mediated Akt activation.
(A) HeLa cells were treated with control, PI3K-C2A, PI3K-C2B or PI3K-C2G siRNA for 48 h. Cells were then infected with wild type S. Typhimurium for 30 min. As controls, cells were uninfected or incubated with 100 ng/mL EGF for 5 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type S. Typhimurium or EGF for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.
Figure 4.
Class III PI3-Kinase does not contribute to SopB-mediated Akt activation.
(A) HeLa cells were treated with control or Vps34 siRNA for 48 h. Cells were infected with wild type or ΔSopB mutant S. Typhimurium for 30 min. As controls, cells were uninfected or incubated with 100 ng/mL EGF for 5 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. Anti-Vps34 antibody was used to verify the knockdown of Vps34 in cells that were treated with siRNA. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD for three separate experiments, calculated as outlined in the Materials and Methods. Statistical significance was assessed by one-way ANOVA analysis.
Figure 5.
IPMK plays a role in SopB-mediated Akt activation.
(A) HeLa cells were treated with control or IPMK siRNA for 48 h. Cells were infected with wild type S. Typhimurium for 30 min. As a control, cells were uninfected. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant S. Typhimurium for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (C) Western blot results of ES10 and ES5 from B were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.