Fig. 3.

Inhibition of Akt phosphorylation on Ser473 reduces the S. aureus internalization by BEC. (A) Cells were left untreated and uninfected (U), were left untreated (−), or were treated with 5 or 10 μM SH-5 for 30 min and then infected with S. aureus at an MOI of 20 for 40 min. A control in which cells were only treated with 10 μM SH-5 for 30 min was also included. After infection, the phosphorylation of Akt was analyzed by Western blotting. Detection of the Akt isoforms 1 to 3 in each sample was performed to ensure equal protein loading. The blot is representative of three independent experiments. (B) The number of internalized S. aureus was analyzed in untreated cells (column 0) or in cells treated with the indicated concentrations of SH-5. Extracellular S. aureus was killed by lysostaphin treatment. (C) The number of adherent S. aureus was analyzed in nontreated cells (column 0) or in cells treated with the indicated concentrations of SH-5. In this case the cells were washed three times with PBS in order to eliminate nonadherent bacteria. Untreated controls contained 0.1% DMSO. Bacteria were recovered from hypotonically lysed cells, cultured on LB agar for 19 to 24 h at 37°C, and counted. The data represent means ± the SEM (n = 3). **, P < 0.001 (compared to the untreated control value).