Increased Cell-Matrix Adhesion upon Constitutive Activation of Rho Proteins by Cytotoxic Necrotizing Factors from E. coli and Y. pseudotuberculosis

Figure 5

Reduced cell migration of adherent cells upon treatment with CNF1 and CNFy. Migration of HeLa cells was analysed in a Boyden chamber experiment. Cell migration was induced by a serum gradient of 10% bovine calf serum for 5 h. Cell migration was quantified as the number of cells that passed bottom membrane. Suspended cells: HeLa cells were pretreated with the indicated toxins for 2 h. Cells were suspended by trypsin, directly seeded onto the membrane and the serum gradient was applied for 5 h. Adherent cells: HeLa cells were seeded on the membrane and allowed to attach. Cells were then treated with the indicated toxins for 2 h. Afterwards the serum gradient was applied for 5 h. Cells on the upper membrane surface were scraped with a cotton swab, and cells on the bottom surface stained with Hoechst 33342. After incubation at 37°C for 15 min, cells were analyzed by fluorescence microscopy using a Zeiss Axiovert 200 M (excitation 365 nm, emission 420 nm). Five adjacent microscope fields for each membrane were counted at ×20 magnification. Results displayed are the mean ± SD of three independent experiments. 𝑃 values <0.05 (*) and <0.01 (**) were considered as statistically significant as compared with nontreated cells.