Influence of simvastatin on hCMEC/D3 confluent and rested monolayers. hCMEC/D3 cells were grown to confluence or further rested for 48 h in medium lacking growth factors. In addition, confluent or rested cultures were incubated with 1 nM Simvastatin overnight at 37°C. A: Immunoblot showing claudin-3 expression under indicated conditions. B-E: Immunolocalization of claudin-3 in confluent (B,C) or rested (D,E) cells in the presence (B,D) or absence (C,E) of simvastatin. Cells were immunostained with an antibody to the claudin-3 protein and visualized with goat anti-rabbit IgG conjugated to Alexa Fluor® 488. Scale bar = 25 μm.