Fig. 6. Vitronectin–vβ3-integrin-mediated invasion of host cells by pneumococci depends on ILK expression. (A) Vitronectin-mediated invasion of pneumococci into ILK-expressing fibroblasts (fl/fl) or ILK^–/– fibroblasts was determined by the antibiotic protection assay. Results are presented as the mean ± s.d. of at least three independent experiments. ^*P<0.004; n.s., non-significant. (B) Immunoblot analysis of ILK expression in ILK-expressing fibroblasts (fl/fl) or ILK-deficient cells (ILK^–/–). (C) Vitronectin-mediated pneumococcal internalization into fibroblasts overexpressing ILK (ILK/ILK) or fibroblasts expressing the N-terminal ankyrin repeats (ILK-ANK). ILK-ANK cells lack the C-terminal kinase domain. Wild-type (Ctrl), ILK^fl/fl (fl/fl) and ILK^–/– fibroblasts were used as controls, and pneumococcal invasion was examined in the presence and absence of the Akt1/2 inhibitor VIII (Akti1/2, 10 µM). Pneumococcal invasion indicates the relative number of intracellular bacteria compared with non-treated fibroblasts (Ctrl). Data are shown as mean ± s.d. of at least three independent experiments. (D) Vitronectin-dependent pneumococcal invasion of non-transfected A549 cells (mock) or cells transfected with the ILK-specific siRNA (ILK-H) or with a control siRNA (Ctrl) was performed 48 hours post-transfection. The number of intracellular and recovered pneumococci was monitored by the antibiotic protection assay. Results are presented as the mean ± s.d. of at least three independent experiments. ^*P<0.004. (E) Reduction of ILK expression was controlled by immunoblot analysis of transfected cell lysates with specific anti-ILK antibodies to verify knock down of ILK by siRNA. Erk1/2 served as loading control. (F, top row) ILK ablation leads to an impaired F-actin cytoskeleton, as demonstrated by immunofluorescence microscopy of ILK-expressing A549 cells (mock) and ILK-knockdown A549 cells (siRNA ILK-H). F-actin is stained with phalloidin and the impact of ILK on the cytoskeleton is also shown for ILK-knockout fibroblasts (ILK^–/–; bottom-right panel). The middle row shows A549 cells treated with vitronectin and infected with pneumococci. Bacteria were stained with Alexa Fluor 488 and Alexa Fluor 568, and appear yellow (green/red stain).