The pEGFP-Rab7 plasmid was generously provided by Dr. Bo van Deurs (University of Copenhagen, Copenhagen, Denmark). Genetic manipulations and sequence verifications were performed following previously described protocols [21,22]. Bacterial and cell culture Bacteria were routinely grown in Millers Luria-Bertani (LB) medium supplemented with antibiotics at 30uC, as indicated. Bacterial growth curves performed in LB in the presence or absence of 100 nM bafilomycin A1, 100 nM wortmannin or 10 mM NH4Cl were monitored in a 96-microwell plate reader BioTek ELx808, with temperature control (37uC), over a 18 h period. No difference in the growth curves of bacteria incubated in the presence of the inhibitors was observed when compared to bacteria grown in LB medium with no additions. Chinese Hamster Ovary (CHO) [23], or the derived stably transfected CHO cells over-expressing EGFP-LC3 (CHO-EGFP- LC3), were grown in a-MEM supplemented with 10% FCS at 37uC and 5% CO2. HeLa [24], or T24 cells [25], wild-type MEF (Mouse Embryonic Fibroblast) and MEF Atg52/2cells (kindly provided by Dr. Tamotsu Yoshimori, Osaka University, Japan) and the derived cells transiently expressing RFP-LC3 were grown in D-MEM supplemented with 10% FCS in the same conditions as CHO cells. Cells were transfected using Lipofectamine 2000 according to the manufacturers instructions. Gentamicin protection assay The gentamicin protection assay was performed as previously described [19] with modifications, as follows. CHO and CHO- GFP-LC3 cells were cultured in 24 wells plates until they reached confluence. S. marcescens were grown without shaking, overnight at 30uC. The bacterial cultures were washed once with PBS and an appropriate volume was added to each well to reach MOI 10. Plates were centrifuged 10 min at 1000 rpm and incubated one hour at 37uC and 5% CO2. Then, the cells were washed with PBS and subsequently incubated with medium supplemented with 10% FCS and 25 mg/ml gentamicin. Cells were then washed four times with PBS and lysed with 0.05% Triton X-100 at indicated time- points. The resultant supernatants were serially diluted and the CFU on LB agar plates were determined. The results for each experiment are the average of an assay performed in triplicate and independently repeated three times. To determine cellular damage, lactose dehydrogenase activity (LDH) in the CHO cell culture supernatant was measured with a Wiener Laboratories S.A. (Argentina) LDH kit in accordance with the manufacturer’s instructions. Percent LDH release was calculated relative to that of the uninfected control, which was set at 0% LDH release, and that of cells lysed in Triton-X 100 (0.05%) treated cells which was set at 100% LDH release. Adherence assay Bacterial adherence assays were performed as described [26]. Briefly, CHO cells were cultured in 24 wells plates until they reached confluence. S. marcescens were grown without shaking, overnight at 30uC. The bacterial cultures were washed once with PBS and the cells were infected (MOI 10). Plates were centrifuged 10 min at 1000 rpm and incubated one hour at 37uC and 5% CO2. The cells were then 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. The results for each experiment are the average of an assay performed in triplicate and independently repeated three times. Serratia Invasion to Epithelial Cells Indirect immunofluorescence and confocal microscopy Infected cells overexpressing EGFP-Rab7 or EGFP-LC3 were fixed with 0.5 ml of 3% paraformaldehyde solution in PBS for 10 min and permeabilized with 0.1% Triton X-100 solution in PBS for 5 min. Subsequently, cells were incubated with primary polyclonal antibodies against S. marcescens (1:500) and detected by incubation with anti-rabbit Cy3 conjugated secondary antibodies (1:150) or anti-rabbit Alexa Fluor 647 conjugated secondary antibodies (1:300). Cells were mounted with SlowFade Antifade reagent in glycerol/PBS (Molecular Probes). Finally, cells were analyzed by confocal microscopy with a Nikon Eclipse TE-2000-E2 and the EZ-C1 3.20 Free Viewer program (Nikon, Japan) or with an Olympus FluoViewTM FV1000 confocal microscope (Olympus, Argentina), and the FV10- ASW (version 01.07.00.16) software, respectively. Colocalization of intracellular bacteria with different markers At different times after infection, cells were fixed for 10 min in 3% paraformaldehyde. Intracellular bacteria were detected by indirect immunofluorescence. At least 200 cells were scored from three independent experiments using confocal microscopy. The percentage of colocalization was calculated as the ratio between the number of vesicles containing bacteria that colocalize with the indicated fluorescently labeled marker and the total number of infected cells. For this purpose, either small vacuoles or large vacuoles filled with bacteria were counted as one vesicle. Detection of degradative compartments with chromogenic DQ-BSA The ability of CHO cells to endocytose and degrade the self- quenched red Bodipy dye conjugated to BSA (DQ-BSA, Molecular Probes) was used to measure degradative lysosomal function. Red DQ-BSA requires enzymatic cleavage in a degradative intracellular compartment to generate a highly fluorescent product which can be monitored by confocal microscopy. CHO-EGFP-LC3 cells were preincubated for four hours at 37uC with DQ-BSA (10 mg/ml in a-MEM supplemented with 10% FCS) to assure that the probe reaches the lysosomal compartment. Subsequently, the cells were infected with S. marcescens as described above and four or six hours post-infection the cells were fixed. Intracellular bacteria were detected by indirect immunofluorescence and the cells were analyzed by confocal microscopy. The percentage of colocalization was calculated as the ratio between the number of vesicles containing bacteria that colocalize with the indicated fluorescently labeled markers and the total number of infected cells. Detection of acidic compartments with LysoTracker CHO-EGFP-LC3 cells were infected with S. marcescens and, at sixty minutes p.i. the acidic compartments were labeled by incubation of infected cells with 3 mM LysoTracker in a-MEM supplemented with 10% FCS at 37uC. Finally, at four or six hours p.i. cells were fixed and intracellular bacteria were detected by indirect immunofluorescence. Cells were analyzed by confocal microscopy. The percentage of colocalization was calculated as the ratio between the number of vesicles containing bacteria that colocalize with the indicated fluorescently labeled markers and the total number of infected cells. Gentamicin protection assay in starvation medium and inhibition by wortmannin. The invasion assays using starvation medium or with the addition of wortmannin (Wn) were performed as the previously PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e24054