described gentamicin protection assay with modifications, as follows. CHO-GFP-LC3 cells were cultured in 24 wells plates until they reached confluence. The cell monolayer was pre- incubated for two hours with medium supplemented with 10% FCS with or without Wn 100 nM or starvation medium EBSS with or without Wn 100 nM. S. marcescens cultures were grown without shaking, ON at 30uC. The bacterial cultures were washed once with PBS and an appropriate volume was added to each well to reach MOI 10, and Wn 100 nM was added where indicated. Plates were centrifuged 10 min at 1000 rpm and incubated one hour at 37uC and 5% CO2. Then, the cells were washed with PBS, incubated with medium supplemented with 10% FCS and 25 mg/ ml gentamicin with the addition of Wn 100 nM where indicated. After 120 or 360 min, cells were washed four times with PBS and lysed with 0.05% Triton X-100. The resultant supernatants were serially diluted and the CFU on LB agar plates were determined. In another set of experiments, Wn 150 nM was added at 60 min p.i. together with gentamicin. At 210 min p.i. the medium was renewed. After 360 min, cells were washed four times with PBS and lysed with 0.05% Triton X-100. The resultant supernatants were serially diluted and the CFU on LB agar plates were determined. Alternatively, cells were mounted and analyzed by confocal microscopy as described above. The results for each experiment are the average of an assay performed in triplicate and independently repeated three times. Gentamicin protection assay with bafilomycin A1 or NH4Cl The invasion assays with the addition of bafilomycin A1 (Baf) or NH4Cl were performed as the previously described gentamicin protection assay with modifications, as follows. CHO-GFP-LC3 cells were cultured in 24 wells plates until they reached confluence. S. marcescens cultures were grown without shaking, ON at 30uC. The bacterial cultures were washed once with PBS and an appropriate volume was added to each well to reach MOI 10, and 100 nM Baf or 10 mM NH4Cl was added. Plates were centrifuged 10 min at 1000 rpm and incubated one hour at 37uC and 5% CO2. Then, the cells were washed with PBS, incubated with medium supplemented with 10% FCS and, 25 mg/ml gentamicin with the addition of 100 nM Baf or 10 mM NH4Cl. After 120, 240 or 360 min, cells were washed four times with PBS and lysed with 0.05% Triton X-100. The resultant supernatants were serially diluted and the CFU on LB agar plates were determined. Alternatively, cells were mounted and analyzed by confocal microscopy as described above. The results for each experiment are the average of an assay performed in triplicate and independently repeated three times. Immunodetection of EGFP-LC3 CHO-EGFP-LC3 cells from the invasion assays where lysed with SDS-PAGE sample buffer. Protein samples from the total cell lysate were run on a 10% polyacrylamide gel (30 mg were loaded per lane) and transferred to Hybond-ECL nitrocellulose mem- branes (GE Healthcare). The membranes were blocked for 2 h with 5% non-fat milk, 0.1% Tris-Buffer Saline (TBS), and washed twice in TBS for 10 min. Then the blots were incubated with primary rabbit anti-GFP polyclonal antibodies, washed twice in TBS and finally incubated with peroxidase-conjugated secondary antibody (GE Healthcare). The blots were developed using an enhanced chemiluminescence detection kit (ECL-Plus) from GE Healthcare. In parallel, the same procedure was carried out to detect the protein a-tubulin, used as loading control. Quantifica- tion of individual bands by densitometry and histograms were performed using the Image J Program. Serratia Invasion to Epithelial Cells Gentamicin protection assay in bacteriostatic conditions CHO-EGFP-LC3 cells were cultured in 24 wells plates until they reached confluence. Overnight S. marcescens cultures were washed once with PBS and incubated one hour at 30uC in LB supplemented with 20 or 100 mg/ml chloramphenicol. Subse- quently, cells were infected (MOI 10) and incubated one hour in a-MEM supplemented with 10% FCS and 20 or 100 mg/ml chloramphenicol. Then, the cells were washed with PBS and medium supplemented with 10% FCS and 25 mg/ml gentamicin was added. After two hours, cells were fixed and intracellular bacteria were detected by indirect immunofluorescence. At least 300 cells were scored from three independent experiments using confocal microscopy. The percentages of invasion and colocaliza- tion with LC3 were calculated as the ratio between the total number of infected cells and the total number of cells, or the number of internalized bacteria that colocalize with LC3 and the total number of cells, respectively. For the assays using heat-killed bacteria, overnight S. marcescens cultures were washed once with PBS and incubated 20 minutes at 65uC. Loss of bacterial viability was verified by determination of CFUs in LB-agar plates. Subsequently, CHO-EGFP-LC3 cells were infected (MOI 10) and incubated one hour in a-MEM supplemented with 10% FCS. Then, an identical protocol to the one described for live bacteria was carried out to determine intracellular CFUs. The results for each experiment are the average of an assay performed in triplicate and independently repeated three times. Statistical analysis Statistical analysis was performed using one-way ANOVA and Holm-Sidak test with an overall significance level = 0.05. Asterisks in the plots denote the values among the treatment groups in which a statistical significant difference was determined. Results Survival and replication of S. marcescens inside epithelial cells Upon infection of CHO epithelial cells, intracellular cumuli of labeled Serratia inside CHO cells were observed by phase contrast and by fluorescence microscopy suggesting that bacteria were enclosed within vacuole-like compartments. The score of the CFUs recovered intracellularly along time showed that Serratia was able not only to survive but also to replicate inside the cells with an estimated 42.8 min doubling time (49 min was the doubling time determined for S. marcescens grown in LB, at 37uC, without agitation), leading to an increase of up to 6-fold in the intracellular counts at 240 min p.i., with no significant decay up to 480 min p.i. (Fig. 1A, representative images of CHO invaded cells at different times p.i. are shown in Fig. 2 and subsequent). Beyond this point, if gentamicin action was eliminated, although CHO cellular integrity was maintained at early times as indicated by monitoring LDH release (see Materials and Methods for details), bacteria slowly started to appear in the extracellular space indicating that they could somehow exit the CHO cells without causing detectable cell damage. If these few extracellular bacteria were not eliminated by restoring gentamicin action, their rapid proliferation caused bacterial numbers that provoked the rounding and detachment of the CHO cells, as well as release of LDH, indicative of cellular damage (equivalent to the results obtained if a MOI higher than 20 was employed in the infection assay, not shown). Further experiments are under way in our laboratory to examine the fate of Serratia and the mechanism that underlies cellular escape at late times p.i. PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e24054