Article Figures & Data
Figures
- Fig 1
Invasion of HCT-8 cells by STEC strains 98NK2 and 98NK2ΔfliC. Invasion assays with either 98NK2 or 98NK2ΔfliC were carried out with (+) or without (−) centrifugation (Spin) for 5 min at 150 × g onto HCT-8 cells. Invasion is expressed as the percentage of total adherent bacteria that were resistant to gentamicin. Data are the means plus SDs (error bars) from three independent experiments, each performed in quadruplicate. Values that are significantly different (P < 0.0001) are indicated by brackets and three asterisks. NS, not significant.
- Fig 2
Association of STEC strains 98NK2 and 98NK2ΔfliC with HCT-8 cells. HCT-8 cells were infected with STEC strain 98NK2 (A) or 98NK2ΔfliC (B) for 1 or 3 h in 8-well chamber slides. Slides were fixed and processed for inside-out immunofluorescence, as described in Materials and Methods. Nuclei are blue (DAPI), extracellular STEC is purple (Alexa Fluor 647), and total STEC is green (Alexa Fluor 488). Bars, 5 μm (top) and 2.5 μm (bottom) (A) and 2.5 μm (B). The white arrow in the merged image in panel B shows colocalized fluorescence indicative of external STEC.
- Fig 3
TLR5 is not involved in invasion of cells by STEC strain 98NK2 or 98NK2ΔfliC. (A) Untransfected HCT-8 cells or cells stably transfected with TLR5-Wt, TLR5-DN, or empty vector (control) were stimulated with strain 98NK2, and RNA was extracted after 4 h. IL-8 mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in the concentration of IL-8 mRNA relative to that in untransfected HCT-8 cells, and data are shown as the means plus SDs for triplicate assays. *, P = 0.0257 compared to control (empty vector) cells or P = 0.0265 compared to cells transfected with TLR5-DN. (B) Invasion assays with STEC strains 98NK2 and 98NK2ΔfliC in HCT-8 cells stably transfected with TLR5-Wt, TLR5-DN, or empty vector. Data shown are the means plus SDs from three independent experiments, each performed in at least duplicate assays (n = 8). NS, not significant. (C) HCT-8 cells were transfected with siRNA directed against TLR5 or with negative-control siRNA, and RNA was extracted 24 h later (as described in Materials and Methods). TLR5 mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in the concentration of TLR5 mRNA relative to control cells. Data shown are the means plus SDs from three independent experiments performed in triplicate. *, P = 0.0106. (D) HCT-8 cells were transfected with siRNA directed against TLR5 or with negative-control siRNA and used in invasion assays performed with strain 98NK2 approximately 48 h later (as described in Materials and Methods). Results shown are the means plus SDs from three independent experiments, each performed in quadruplicate. NS, not significant.
- Fig 4
MyD88 is not involved in invasion of cells by STEC strain 98NK2 or 98NK2ΔfliC. (A) Invasion assays with STEC strains 98NK2 and 98NK2ΔfliC in HCT-8 cells transiently transfected with MyD88-DN, IRAK-Wt, IRAK-Wt, TLR5-DN, or TLR5-Wt approximately 24 to 36 h prior to invasion assays (as described in Materials and Methods). Results shown are the means plus SDs from three independent experiments in duplicate wells. Significant differences are indicated as follows: *, P values ranged from 0.0002 to 0.0494; NS, not significant. (B) HCT-8 cells were transfected with siRNA directed against MyD88 or with negative-control siRNA, and RNA was extracted 24 h later (as described in Materials and Methods). MyD88 mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in the concentration of MyD88 mRNA relative to control cells. Data shown are the means plus SDs from three independent experiments performed in triplicate. Significant differences are indicated as follows: **, P = 0.0028. (C) HCT-8 cells were transfected with siRNA directed against MyD88 or with negative-control siRNA and used in invasion assays performed with strain 98NK2 approximately 48 h later (as described in Materials and Methods). Results shown are the means plus SDs from three independent experiments, each performed in quadruplicate. NS, not significant.
- Fig 5
Role of asialo-GM1 and neuraminidase in invasion of HCT-8 cells by STEC strains 98NK2 and 98NK2ΔfliC. (A) HCT-8 cells were pretreated with anti-asialo-GM1 antibody (1:100) or normal rabbit serum (control) for 1 h prior to infection. Invasion assays were then performed with STEC strain 98NK2 or 98NK2ΔfliC. Results shown are the means plus SDs from five independent experiments, each performed in triplicate. **, P = 0.0093; NS, not significant. (B) HCT-8 cells were pretreated with 2.5 U/ml neuraminidase or with vehicle control (PBS containing 25 mM KCl [pH 6.0]) for 1 h prior to infection. Invasion assays were then performed with 98NK2 or 98NK2ΔfliC. Results shown are the means plus SDs from three independent experiments, each performed at least in triplicate. **, P = 0.0051; *, P = 0.0115.
- Fig 6
Role of genistein and MβCD in invasion of HCT-8 cells by STEC strains 98NK2 and 98NK2ΔfliC. (A) HCT-8 cells were pretreated with 100 μg/ml genistein or vehicle control (water) for 1 h prior to infection. Invasion assays were then performed with STEC strain 98NK2 or 98NK2ΔfliC. Results shown are the means plus SDs from three independent experiments in triplicate. **, P = 0.0056; *, P = 0.0459. (B) HCT-8 cells were pretreated with either 5 or 10 mM MβCD or with vehicle control (DMSO) for 1 h prior to infection. Invasion assays were then performed with strain 98NK2 or 98NK2ΔfliC. Results shown are the means plus SDs from three independent experiments in triplicate. ***, P < 0.0001; **, P = 0.0002; *, P = 0.0456; NS, not significant.
- Fig 7
Association of STEC strain 98NK2 with the lipid raft marker caveolin-1. (A) Uninfected HCT-8 cells in chamber slides were fixed and processed for caveolin-1 labeling as described in Materials and Methods. (B and C) HCT-8 cells were infected with strain 98NK2 for 1 h (B) or 3 h (C) in chamber slides. Slides were fixed and processed for inside-out immunofluorescence and caveolin-1 labeling as described in Materials and Methods. Nuclei are blue (DAPI), extracellular STEC is purple (Alexa Fluor 647), total STEC is green (Alexa Fluor 488), and caveolin-1 is red (Alexa Fluor 594). Bars, 2.5 μm (A and C) and 5 μm (B).
- Fig 8
Association of STEC strain 98NK2 with the lipid raft marker GM1. (A) Uninfected HCT-8 cells in chamber slides were incubated for 1 h at 37°C in the presence of CtxB-AF594 and then fixed and processed as described in Materials and Methods. Bar, 2.5 μm. (B and C) HCT-8 cells were infected with 98NK2 for 2 h (B) or 3 h (C) in chamber slides and also incubated with CtxB-AF594 (red) for 1 h at 37°C. Slides were then fixed and processed for inside-out immunofluorescence as described in Materials and Methods. Nuclei are blue (DAPI), extracellular STEC is purple (Alexa Fluor 647), and total STEC is green (Alexa Fluor 488). Bars, 2.5 μm.
