Vps34 expression is required for PI(3)P formation on the SCV. (A) Western blot analysis of HeLa cell lysates. Tubulin expression was assessed as a loading control. (B–E) Cells were treated with control (B and D) or Vps34 (C and E) siRNA before transfection with either PH(Akt)-GFP (B and C) or 2FYVE-GFP (D and E). Cells were then infected with WT bacteria (Salmonella-RFP) and analyzed by confocal microscopy. Insets in D and E are enlarged from dashed boxes. These images correspond to <15 min of infection. Bars, 10 μm. (F) Cells were treated with control or Vps34 siRNA and infected with WT bacteria expressing RFP. Infected cells were analyzed using confocal microscopy. Images were acquired at 1-min intervals for ∼1 h. Colocalization of the bacteria with 2FYVE-GFP during infection is shown. Data are means ± SEM of four separate experiments for control siRNA–treated cells (102 SCVs analyzed) and three separate experiments for Vps34 siRNA–treated cells (170 SCVs analyzed). The p-value is shown.