Figure 2.

Rab9 does not regulate Rab32 recruitment to the Salmonella-containing vacuoles (SCV) and its function is not required for Rab32/BLOC-3 dependent killing. (A) GFP-Rab9 transfected HeLa cells were infected with mCherry S. Typhimurium ΔgtgEΔsopD2 (ΔΔ) or WT for 2.5 h, fixed and images were acquired using fluorescent microscopy. (B) The percentage of S. Typhimurium in Rab9-positive vacuoles at 2.5 h post-infection is shown with ± standard-deviation of three independent experiments. (C) HeLa cells transfected with either GFP or GFP-Rab9 were infected with mCherry S. Typhimurium WT for 2.5 h. Cells were then fixed, stained with monoclonal anti-HPS4 (gray) antibody and analyzed by fluorescence microscopy. (D) The percentage of S. Typhimurium in HPS4-positive vacuoles at 2.5 h post-infection is shown with ± standard-deviation of three independent experiments. (E) iBMDM cells knocked down for Rab9 (Rab9 KD) and control (Scrmbl) were infected with mCherry S. Typhimurium ΔgtgEΔsopD2 or WT for 2.5 h, fixed and stained with anti-Rab32 (green). Images were acquired using fluorescent microscopy (F) The percentage of S. Typhimurium in Rab32-positive vacuoles is shown with the ± standard-deviation from three independent experiment. (G) iBMDM cells depleted for Rab9 (Rab9 KD) and control cells (Scrmbl) were infected with S. Typhimurium ΔgtgEΔsopD2 or WT, lysed at the indicated time points and colony-forming units were calculated. Values are presented as fold change compared to the initially internalized (1.5 hpi). Error bars indicate standard-deviation of five independent experiments. For the non-normalized values see Figure S2 . (Student’s t test; *p < 0.05, **p < 0.01) S. Tm, Salmonella Typhimurium; KD, Knock-down; CFUs, Colony-forming units; Scale bar: 5μm; NS, non statistically different.