601 ± 0.115) compared to that of E22 WT infection. On the contrary, E22ΔfliC infection produced lower selleck chemical ERK1/2 phosphorylation (0.681 ± 0.104) than E22 WT infection. These results

confirmed that flagellin is necessary for full ERK1/2 phosphorylation, but it also indicates that intimin has the opposite effect and works as a negative modulator of ERK1/2. To detect ERK1/2 nuclear translocation, a crucial phase in the activation of this pathway, cells infected by EPEC were analysed by immunofluorescence and confocal microscopy using antibodies against ERK1/2 (Fig. 3). FBS (a positive control) caused ERK1/2 nuclear translocation, detected as an intense ERK1/2 signal inside the cell nucleus (green signal into the nucleus). In mock-infected cells, as well as in HB101

stimulated HM781-36B concentration cells, ERK1/2 was restricted to the cytoplasm outside the nucleus. In contrast, in cells infected with EPEC strains (E22 or E2348/69) ERK1/2 was localized in the nuclear compartment (Fig. 3). The intensity and distribution of ERK1/2 in EPEC-infected cells was similar to the patterns observed in FBS-treated cells. These experiments showed that EPEC infection promotes ERK1/2 phosphorylation and induces its nuclear translocation. To understand the role of EPEC virulence in ERK1/2 nuclear translocation, ERK1/2 subcellular localization was tested in cells infected with E22 Δeae, ΔescN, ΔespA and ΔfliC isogenic mutants (Fig. 4). The presence of ERK1/2 inside the nuclei was lower in cells infected with mutants in intimin, flagellin and the T3SS (in the latter it was almost abolished), in comparison Loperamide with the intense mark for ERK1/2 inside the nuclei of E22 WT-infected cells (Fig. 4). These results indicate that ERK1/2 nuclear

translocation during EPEC infection requires the presence of flagellin and needs translocation of effectors by T3SS, and intimate adherence. NF-κB is a crucial proinflammatory pathway activated by EPEC. To analyse NF-κB activation, we measured the phosphorylation and degradation of its inhibitor (IκB-α). By flow cytometry, we quantified IκB-α in cells that interacted with HB101 or were infected with EPEC strains E2348/69, E22 WT or E22ΔfliC for 2 h (Fig. 5A) or 4 h (Fig. 5B). Most of the mock-infected cells (67%) were positive for IκB-α; however, in a fraction of the cell population (33%), IκB-α levels were similar to those detected in the FITC-control. This result could reveal IκB-α basal degradation in HT-29 cells. Cells treated with HB101 did not have less IκB-α than mock-infected cells (average fluorescence value of 18.3 ± 0.6), and no significant differences were detected at 2 (17.5 ± 0.8) or 4 h (17.4 ± 1.4) (Fig. 5A, B). However, cells infected with E2348/69 showed lower levels of IκB-α (14.9 ± 1.3 at 2 h and 11.3 ± 1.9 at 4 h of infection) in comparison with mock-treated cells. E22 WT infection did not significantly change IκB-α levels at 2 h of infection (17.5 ± 2.