NleL Modulates Pedestal Formation Figure 4. The E3 ligase activity of EHEC NleL down modulates EPEC pedestal formation. HeLa cells were infected at a multiplicity of infection of 100 with wild type EPEC, or EPEC harboring plasmid expressing wild-type EHEC NleL or NleLC753A. Cells were infected for 4 hours. (A) Bacteria were visualized by staining with an anti-EPEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (B) The number of micro-clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–10 pedestals, 11–20 pedestals and .20 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars. doi:10.1371/journal.pone.0019331.g004 Bacterial infection EHEC strains were grown overnight standing at 37uC in Luria- Bertani (LB) broth supplemented with nalidixic acid (50 mg ml21) and used directly for infection [20]. EPEC strains were grown overnight standing at 37uC in LB supplemented with followed by subculture (1:100) in Hepes-buffered DMEM until optical density of the cultures measured at 600 nm reached 0.7 [9]. Cultures were then used to infect HeLa cells grown in 24-well tissue culture plates at 37uC in 5% CO2 with 10% fetal calf serum (Gibco, Carlsbad, CA, USA), at a multiplicity of infection (moi) of 100, unless specified otherwise. PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e19331