Serratia Invasion to Epithelial Cells Figure 8. Confocal microscopy analysis of the induction or inhibition of autophagy on Serratia invasion in CHO cells. CHO-EGFP-LC3 cells subjected to the invasion assays as described in Fig. 5B were visualized by confocal laser microscopy. Bacteria were detected with antibodies anti-Serratia coupled with a secondary antibody labeled with Cy3. Representative DIC (a and e), EGFP-LC3 green fluorescence (b and f), red fluorescence labeled bacteria (c and g) and merged fluorescence images (d and h) are shown. In the assays where wortmannin was used, representative images from both the scarce SeCV observed (a-d) and from barely invaded cell fields (e-h) are shown. The lower panel shows representative images of control, non-invaded cells incubated with a-MEM, in starvation medium or in starvation medium + Wn (green EGFP-LC3 fluorescence, a, b and c, respectively). Bars: 10 mm. The percentage of invaded cells displaying the phenotype shown in the images in the corresponding row is indicated to the right. At least 200 cells were counted for each condition; the media 6 S.D. is shown for two independent experiments. doi:10.1371/journal.pone.0024054.g008 epithelial CHO cells and examined events that determine the fate of the pathogen inside a non-phagocytic cell type. In the present report we demonstrate that Serratia is able not only to be internalized but also to replicate within different epithelial cell lines. Once inside CHO cells, bacteria augmented 6-fold its population in the 2 to 4 h p.i. lapse. Afterwards, intracellular counts remained essentially stable up to 8 h p.i. The steady state in bacterial CFUs reached by Serratia beyond 4 h p.i. could be interpreted as equilibrium reached between bacterial propagation and cellular elimination mechanisms or to a modulation of bacterial division controlled by bacteria-cell interaction cues. Correlating with these results, small bacteria-containing vacuoles (1–4 bacteria/ PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e24054