Serratia Invasion to Epithelial Cells Figure 2. LC3 and Rab7 are recruited to the S. marcescens-containing vacuole. A) CHO-EGFP-LC3 cells (green fluorescence) or B) CHO cells transfected with EGFP-Rab7 (green fluorescence), were infected with wild-type S. marcescens and cells were fixed at 120 min p.i (a-d) or 360 minutes p.i (e-h). Bacteria were detected with antibodies anti-Serratia coupled with a secondary antibody labeled with Cy3 (red fluorescence). Representative confocal Z-slices are shown. Inset images show higher magnification of the boxed areas in merged images. Bars: 10 mm. C) CHO-EGFP-LC3 cells or CHO cells transfected with pEGFP-Rab7 were infected with wild-type S. marcescens. Cells were fixed at the indicated time points and bacteria were detected with antibodies anti-Serratia coupled with a secondary antibody labeled with Cy3. The percentage of colocalization of EGFP-LC3 (N) or EGFP-Rab7 (m) with bacteria was determined by fluorescence microscopy. At least 200 infected cells were counted for each time point. The average 6 S.D. for three independent experiments is shown. doi:10.1371/journal.pone.0024054.g002 We simultaneously evaluated if LC3-labeled SeCVs displayed features of degradative compartments. With this aim, DQ-BSA was preloaded into GFP-LC3-expressing CHO cells 4 hours previous to infection. DQ-BSA is a self-quenched fluorophore that emits red fluorescence upon proteolytic digestion, useful to monitor degrada- tive vacuoles. In this assay, at 240 min p.i., co-localization of SeCVs and LC3 was estimated in 66.6%., while 53.9% SeCV were LC3-labeled vesicles and did not show co-localization with DQ- BSA. 18.1% SeCVs were labeled by the degradative marker but not by LC3 (quantitation of the Serratia-containing vesicles colocaliza- tion relative to total infected cells with EGFP-LC3 and DQ-BSA assay at 240 and 360 min p.i. is given in Fig. 4C, representative captured images from 360 min p.i., indicating both degradative - arrows- and non-degradative -inset- vacuoles are shown in Fig. 4D). PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e24054