5% paraformaldehyde, and lysed in 1% Triton X-100 for 5 min at room temperature. Monolayers were then Selleck PLX-4720 washed three times, incubated in a dark chamber with 5 μg/mL phalloidin

(20 min), and washed. Coverslips were mounted in glycerol with 0.1% para-phenylenediamine to reduce bleaching. Transmission Electron Microscopy T84 cells were cultured in Transwell membranes (Costar) for 14 days and infected as described above. Then they were washed 3 times (10 min each) with D-PBS (Sigma) and fixed with 2% glutaraldehyde (Serva) for 24 h at 4°C. After fixation, cells were washed 3 times with D-PBS (10 min) and post-fixed with 1% osmium tetroxide RGFP966 (Plano). Cells were dehydrated through a graded ethanol series (30%, 50% and 70%), then filters were cut out from the cell culture system holder and preparations were treated with ethanol (90%, 96% and 99.8%), followed by propylenoxid (100%), Epon:Propylenoxid (1:1, Serva), and Epon 100%. Afterward, filters were embedded in flat plates

and kept for 2 days for polymerization. Ultrathin sections were prepared, stained with 4% uranyl acetate (Merck) and Reynold’s lead citrate (Merck), and were examined with a Tecnai G2 Spirit Twin, Fei Company at 80 kV. Alternatively, ARN-509 price T84 cells were cultured on 35 mm diameter plates for 14 days. Infection, fixation and dehydration were performed as described above. Subsequently, the cells were examined with a LEO 906E transmission electron microscope (Zeiss, Germany) at 80 kV. Statistical analyses Differences in the percentages of invasion were assessed for significance Cisplatin concentration by using an unpaired, two-tailed t test (GraphPad Prism 4.0). Acknowledgements Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 08/53812-4), and Programa de Apoio a Núcleos de Excelência – PRONEX MCT/CNPq/FAPERJ supported this work. DY received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, fellowship 141708/04); DY and RTH received sandwich fellowships from

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Programa Brasil Alemanha (CAPES – Probral 281/07). Additional funding of this work was obtained from DAAD PPP-Brasilien (D/06/33942) and the European Network ERA-NET PathoGenoMics (Project 0313937C) and from Spanish Ministry of Health and Consumer Affairs (Fondo de Investigación Sanitaria, Spanish Network for the Research in Infectious Diseases, REIPI, RD06/0008-1018), Spanish Ministry of Education and Science (AGL-2008-02129) and the Autonomous Government of Galicia (Xunta de Galicia, PGIDIT065TAL26101P, 07MRU036261PR). A. Mora acknowledges the Ramón y Cajal programme from The Spanish Ministry of Education and Science. We also thank Dr. Cecilia Mari Abe for her help in some of the TEM procedures and J.R.C. Andrade for donating the Salmonella enterica serovar Typhimurium control strain. References 1. Kaper JB: Defining EPEC. Rev Microbiol 1996, 27:130–133. 2.