<i>Legionella</i>-induced phosphorylation of rS6p requires MTOR and PI3K activity.

<p><b>(a)</b> Simplified schematics of the PI3K/MTOR signaling axis indicating the inhibitors used in this study. PI3K is inhibited by LY294002. Rapamycin, PP242 and Torin2 act on MTOR and Hanks' Balanced Salt Solution (HBSS) blocks MTOR by starving cells for amino acids. <b>(b-d)</b> Analyses of serum-starved <i>Myd88</i>^-/- BMMs unstimulated or infected with <i>ΔflaA</i> (MOI = 20) for 5hrs under serum-free conditions. Inhibitors—Rapamycin (200nM), PP242 (5μM), LY294002 (10μM)—or HBSS were added at the time of infection synchronization at 60 min post infection. <b>(b)</b> Immunoblot analysis of S6K1 and rS6p phosphorylation from cell lysates showing quantified band intensities normalized to uninfected conditions (UN). <b>(c)</b> Single cell immunofluorescence analysis of phospho-rS6 positive (MFI>300) <i>Myd88</i>^-/-macrophages exposed to <i>ΔflaA</i> (MOI = 20). Graphed are the means and standard deviations (s.d) of technical triplicates for the two distinct groups within the cell population—infected (LCV present) and uninfected (LCV absent) for each condition. At least 100 cells were analyzed for each condition. ** p<0.005 (one-way ANOVA) <b>(d)</b> Immunofluorescense micrographs of representative infected cells from each condition stained with anti-<i>L</i>. <i>pneumophila</i> (L.p), anti-p-rS6p (S235/236), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate <i>Legionella</i>-containing vacuoles, Bar = 5μm. <b>(b-d)</b> A representative of three biological replicates is shown for each experiment.