18 ± 2.55% , while in 3-MA NVP-LDE225 ic50 or Wm pretreated cells was approximately 10.95 ± 2.65% and 9.39 ± 2.78%, respectively (Figure 6B). Figure 6 Inhibition of autophagy by pharmacological inhibitors reduced the co-localization of E. coli with autophagosomes. (A) HMrSV5 cells were infected with fluorescent E. coli (green) for 1 hour. Following phagocytosis, HMrSV5

cells were exposed for 12 hours in control condition, LPS (1.0 μg/ml), 3-MA (10 mM), Wm (50 nM), LPS + 3-MA or LPS + Wm. Cells were labeled with MDC (blue) for the detection of autophagic vacuoles formation. Scale bars: 20 μm. (B) Quantitation of the co-localization of E. coli with the MDC-labeled autophagosomes in Figure 6A (mean values ± SD, n ≥ 3). ** p < 0.01 (vs. control); # p < 0.05 (vs. LPS). Downregulation

of autophagy by Beclin-1 siRNA reduced LPS-induced bactericidal activity and the co-localization of E. coli with autophagosomes To more specifically determine whether LPS-induced antimicrobial activity was dependent on autophagy, short interfering RNA (siRNA) specific for Beclin-1 was used to transfect the HMrSV5 cells and block autophagic responses. Figure 7A shows that knockdown of Beclin-1 effectively reduced expression of Beclin-1 and LC3-II protein. Meanwhile, fewer autophagic vacuoles labeled by MDC were FK866 datasheet observed in HMrSV5 cells transfected with Beclin-1 siRNA (Figure 7B and C). Figure 7 LPS-induced bactericidal activity was attenuated after deletion of Beclin-1 by siRNA in HMrSV5 cells. After transiently transfected with negative control siRNA or Beclin-1 siRNA, the HMrSV5 cells were incubated with LPS (1.0 μg/ml) for 12 hours. (A) The left panel shows representative western blots probed with antibodies against Beclin-1 and LC3-II. The right panel shows densitometric analysis of Beclin-1 and LC3-II in the left panel;

β-actin was used as a loading control. (B) After transfection, MDC-labeled autophagic vacuoles were observed. Scale bars: 20 μm. (C) Quantitation of the number of MDC-labeled autophagosomes per cell in Figure 7B. * p < 0.05 in Figure 7A and 7C U0126 in vivo (vs. control); # p < 0.05 in Figure 7A and 7C (vs. LPS). (D) Graph represents percentage of remaining E.coli at different time points in each group treated as described above. Data are mean values ± SD (n ≥3). * and ** denote p < 0.05 and p < 0.01 respectively (LPS vs. control); # denote p < 0.05 (LPS + Beclin-1 siRNA vs. LPS). We subsequently examined the bactericidal activity of the siRNA-transfected cells in response to E. coli. Compared with control cells incubated with LPS alone, loss of Beclin-1 in HMrSV5 cells markedly attenuated bactericidal activity induced by LPS (Figure 7D). In addition, we further used MDC staining to look for E. coli-targeted autophagosomes. Consistent with the pharmacological inhibition of autophagy by 3-MA and Wm, co-localization of E. coli with MDC-labeled autophagosomes decreased from 28.98 ± 4.23% to 12.88 ± 2.34% (p < 0.