Electron microscopy of coculture experiments and real-time RT-PCR analysis. (A) Electron microscopy of confluent Caco-2 cells (I) cocultured with E. coli MG1655 (II) or EcN (III) was performed in order to visualize the interaction between cells and bacteria. (B) Validation of mRNA expression levels of Caco-2 cells by quantitative real-time RT-PCR. After 6 hours of coincubation with either E. coli MG1655 (+ K12) or EcN (+ EcN), total RNA was isolated, reversely transcribed, and relative mRNA expression levels for selected genes and the housekeeping gene RPS-9 (as internal control) were analyzed in duplicate real-time RT-PCR assays. Relative mRNA amounts were normalized with respect to expression levels of untreated Caco-2 cells (fold change = 1). The figure is representative of two independent experiments. MCP-1 indicates chemoattractant protein-1 ligand 2; MIP-2α, macrophage inflammatory protein-2 alpha; MIP-2β, macrophage inflammatory protein-2 beta; DUSP5, dual specificity phosphatase 5; NFκBIA, nuclear factor of kappa light polypeptide gene enhancer in B cell inhibitor, alpha; TNFαIP3, tumor necrosis factor, alpha-induced protein 3; VEGF, vascular growth factor and ELF3, E74-like factor 3 (ets domain transcription factor, epithelial-specific).