Figure 4

Knock-down of vinculin in wildtype fibroblasts does not affect S. aureus uptake. (A) Murine fibroblasts were transduced with lentiviral particles harboring either pLKO.1 or pLKO.1 encoding shRNA against mouse vinculin (pLKO-1 sh-mvinc). WCL of the stably transduced cell lines were analyzed for FAK, vinculin, paxillin, Src and β-tubulin protein expression by Western blotting. Representative blots are shown. (B) Integrin α5 surface expression in cells from (A) was analyzed by flow cytometry. Shown is a representative histogram of cells stained with α-integrin α5 antibodies and a rhodamine-coupled secondary antibody (blue – pLKO.1; red – pLKO.1 sh-mvinc) or cells stained with the secondary antibody only (contr.; grey lines). (C) pLKO.1 or pLKO.1 sh-mvinc cells were seeded on gelatine coated 24 well plates. Next day, cells were infected with S. aureus at MOI 20 for 2 h. Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (top) or gentamicin protection assays (bottom), respectively. Values are means ± SEM from 2 independent experiments done in quadruplicate, n=8. Mann-Whitney test was applied for statistical testing.