Serratia Invasion to Epithelial Cells Figure 6. Analysis of LC3-I/LC3-II conversion. CHO-EGFP-LC3 cell monolayers subjected to the invasion assays as described in Figure 5 were harvested and resuspended in SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE (30 mg of total protein was loaded per lane), transferred to nitrocellulose and detected with anti-GFP polyclonal antibodies (‘‘invasion’’, lower panel blots). Control, non- invaded CHO-EGFP-LC3 cells, that were subjected to the same treatments were also analyzed (‘‘control’’, upper panel blots). The positions of LC3-I and LC3-II bands are indicated. Protein loading was controlled according to the a-tubulin content (the blots detected with antibodies anti-a-tubulin are shown). Blots are representative of three independent experiments. The bands intensity was quantified with the ImageJ Programe, and the LC3II/LC3I (II/I) was calculated for the immunodetection experiment shown in the figure. The histogram represents the average 6 S.D. of the relative intensities of LC3-I or LC3-II versus a-tubulin of three independent experiments. doi:10.1371/journal.pone.0024054.g006 observed in both invaded and non-invaded cells 120 min p.i (Fig. 10B, a–d). Interestingly, LC3 puncta was replaced by a cytoplasmic homogeneous fluorescence only in non-invaded cells, provided that extracellular bacteria were eliminated by the action of gentamicin (Fig. 10B, e–h). This observation also correlates with our previous result showing that bacterial de novo protein synthesis was required to induce the autophagy phenotype in CHO cells (Fig. 9B and C). These results indicate that the autophagy process is neither induced by flagellar-dependent adhesion of Serratia to the host cell nor by the expression of PhlA. They also reinforce the notion that autophagy could be activated by the bacteria from Figure 7. Confocal microscopy analysis of the effect of altering vacuolar acidification on Serratia invasion. CHO-EGFP-LC3 cells subjected to the invasion assays as described in Fig. 5A were visualized by confocal laser microscopy. a and b panels show representative images of control, non-invaded cells and c-f panels depict invaded cells incubated with a-MEM, a-MEM +100 nM Baf or a-MEM +10 mM NH4Cl, as indicated. Bacteria were detected with antibodies anti-Serratia coupled with a secondary antibody labeled with Cy3. Representative DIC (c), EGFP-LC3 green fluorescence (d), red fluorescence-labeled bacteria (e) and merged images (f) are shown. At least 300 cells were inspected for each condition in two independent experiments. Bars: 10 mm. doi:10.1371/journal.pone.0024054.g007 outside the target cell, being the autophagic phenotype sustained at later stages post-infection only in invaded cells. Discussion To understand the mechanisms that Serratia uses to invade and establish in the host, we set up an in vitro infection assay into PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e24054