Transient vinculin overexpression in 293 cells does not interfere with bacterial uptake. (A) 293 cells were transfected with constructs encoding GFP or GFP-vinculin. 48 h post-transfection whole cell lysates (WCLs) were prepared and analyzed for vinculin and β-tubulin expression by immunoblotting. Representative blots are shown. (B) Cells transfected as in (A) were compared for integrin α5 surface expression by flow cytometry. Shown is a representative histogram of cells stained with α-integrin α5 antibody and a rhodamine-coupled secondary antibody (blue – GFP; red – GFP-vinculin) or cells stained with the secondary antibody only (grey lines). (C and D) Cells as in (A) were infected with S. aureus and S. carnosus for 2 h (MOI 20). Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (C) or gentamicin protection assays (D), respectively. Values are means ± SEM from 2 independent experiments done in quadruplicate, n=8. Mann-Whitney test was applied for statistical significance testing.