The absorbance values for the enzymatic reactions at 490 nm were registered in an ELISA Microplate Reader 550 (Bio-Rad). Data were charted (absorbance

versus concentration) and https://www.selleckchem.com/products/epacadostat-incb024360.html analysed to construct lineal regression equations for determining the concentrations of each cytokine. To quantify the secreted cytokines in our infection model, HT-29 cells (1 × 106) disposed on 35 × 10 mm culture dishes were used for bacterial interaction. Supernatants (200 μl for each condition) were collected, mixed 1:1 with 2× coating buffer (final concentration 1×) and adsorbed overnight at 4 °C. ELISA determination was developed as described for the standard curves, and absorbance values were used to calculate IL-1β, IL-8 or TNF-α concentrations in the supernatants. Statistical analysis.  All numerical data are presented as the mean and standard deviation (SD) for at least three independent experiments. Data comparisons were made using the Student’s t-test. A P value <0.05 was considered statistically significant. We wanted to define if TLR5 is expressed in HT-29 intestinal epithelial cells and analyse if its expression is modified by EPEC infection. Analysis by RT-PCR indicated that

HT-29 cells expressed tlr5 mRNA (RT-PCR product normalized intensity Selleck ZVADFMK of 0.721 ± 0.202). The expression of tlr5 was not altered by cell interaction with non-pathogenic E. coli HB101 or by infection with EPEC strains E2348/69 or E22 (Figure S1). We also analysed the possible influence of EPEC intimin, T3SS and flagellin over tlr5 expression. As with E22 wild-type, infection with any E22 isogenic mutants did not change tlr5 mRNA expression (Figure S1). These data suggest that tlr5 expression in HT-29 cells is not modified during EPEC infection.

We also explored TLR5 protein expression by WB assays. We found that HT-29 cells expressed TLR5, which was easily detected, and the quantity of TLR5 was not altered Thiamine-diphosphate kinase by interaction with the E. coli strains HB101, E2348/69, E22 WT, E22Δeae, E22ΔescN, E22ΔespA or E22ΔfliC (Figure S1). These data indicate that HT-29 intestinal epithelial cells express TLR5 protein constitutively, and its expression is neither altered during interaction with non-pathogenic E. coli nor during infection with EPEC wild-type strains or E22 mutants in intimin, T3SS components or flagellin-encoding genes. Toll-like receptors are not restricted to the cell membrane and can be retrieved from intracellular vesicles [38]. To analyse the subcellular localization of TLR5 in EPEC-infected HT-29 intestinal epithelial cells, we performed flow cytometry assays to detect and compare total TLR5 (FACS of permeabilized cells) and TLR5 on the cell surface (FACS of non-permeabilized cells).