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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Cell Microbiol. 2010 Jan 20;12(7):919–929. doi: 10.1111/j.1462-5822.2010.01440.x

Figure 5. Inhibitors of CME and lipid rafts block EPEC invasion but not EPEC-induced TER loss.

Figure 5

A. Gentamicin protection assays in Caco-2 cells infected with wild-type EPEC suspended in serum-free DMEM (Untreated) or serum-free DMEM with 0.1% DMSO, 60μM chlorpromazine (CPZ), 80μM dynasore, 10mM methyl-beta-cyclodextrin (MBCD), or 400μM monodansyl cadaverine (MDC). Invasion was significantly inhibited in cells treated with CPZ, MBCD, or MDC. *: p<0.05. Bacterial attachment was not affected by inhibitor treatment (data not shown). Relative invasion was calculated as described in Methods and WT normalized to 1.0. B. GPAs performed as in A but with Salmonella in the presence of media alone (Untreated), 0.1% DMSO or 80μM dynasore. Dynasore treatment significantly reduced the rate of Salmonella invasion. *: p<0.05. C. Caco-2 cells were grown on permeable supports for 14 days and transepithelial electrical resistance (TER) was measured after treatment with serum-free DMEM (Uninfected) or serum-free DMEM with wild-type EPEC (EPEC) for 6 hours. TER was significantly reduced by EPEC infection in the presence of media alone (Untreated) or media with 0.1% DMSO, 60μM chlorpromazine (CPZ), 10mM methyl-beta-cyclodextrin (MBCD), or 400μM monodansyl cadaverine (MDC). Inhibitor treatment alone did not reduce TER. D. HeLa cells transiently transfected with either EGFP-EspF (EspF), YFP-clathrin light chain (Clathrin) or EGFP-dynamin II (Dynamin) were infected for 2.5 hours with wild-type EPEC and processed for epifluorescent microscopy. All three fluorescent fusion proteins were found to accumulate around attached bacteria, shown by DAPI staining (EspF, Clathrin) or by phase contrast (Dynamin).