amounts of proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Protein bands were transferred electrophoretically onto Immobilon-P polyviny- lidene difluoride membranes (Millipore, MA, USA). The mem- branes were blocked with 5% fat-skim milk in TBS (Tris-buffered saline; pH 7.5), containing 0.05% Tween-20 for 1 h at RT. Next, membranes were incubated with the monoclonal mouse anti-LC3 (overnight, at 4uC) or anti-b-actin (1 h at RT) diluted in TBS- 0.05% Tween-20. Membranes were washed and then incubated with secondary antibodies conjugated with horseradish peroxidase. Signal detection was performed with the enhanced chemilumi- nescence system (ECL, Amersham). Inhibition of lysosomal acidification MEFs were seeded onto coverslips and exposed to IFNc for 23 h. MEFs were pretreated with 100 nM Baf A1 for 1 h before the infection. Cells were then inoculated with C. trachomatis (MOI of 5), and continuously incubated with Baf A1 throughout the experiment period. Control cell monolayers were treated with dimethylsulfoxide, in which Baf A1 was dissolved. Specimens were then fixed and double stained for LAMP1 and C. trachomatis. The percentages of colocalization of chlamydial inclusions with LAMP1 were determined in the presence or absence of the inhibitor. To examine the effect of the acidification inhibition on the recovery of infectious chlamydial progeny, cells were grown in 6- well-plates, treated, and infected as mentioned above. Cells were then harvested 2 days p.i., lysed and diluted, and formation of infectious C. trachomatis was assessed by infectivity titration assays on fresh HeLa cell cultures. Assessment of host cell viability Lactate dehydrogenase (LDH) colorimetric assay (Roche Diagnostics, Mannheim, Germany) was used according to manufacturer’s instructions. This assay is based on the measure- ment of LDH activity released from the cytosol of damaged cells into the supernatant. Supporting Information Figure S1 No colocalization of Irga6, Irgb6, Irgd, Irgm1, Irgm2 and Irgm3 at early C. muridarum inclusions upon IFNc stimulation. Double immunofluorescence labelling of IRGs and C. muridarum in WT (A), Atg5 2/2 (B) and Irga62/2 (C) MEFs stimulated for 24 h with 100 U/ml IFNc and then infected for 3 h with C. muridarum (MOI 5). IFNc untreated MEFs were similarly infected. Upon IFNc induction all tested IRGs except Irgb6 were highly expressed in all cell lines (no Irga6 in Irga6 2/2 MEFs) without any colocalization to bacterial inclusions. Scale bar represents 5 mm Found at: doi:10.1371/journal.pone.0004588.s001 (2.84 MB TIF) Figure S2 IFNc cannot induce autolysosomal fusion with early C. muridarum inclusions to suppress bacterial growth in WT, Atg52/2 and Irg62/2 MEFs. (A) C. muridarum inclusions avoid interaction with lysosomes in all MEFs tested despite IFNc induction. IFNc treated and untreated Irga6, Atg52/2 and WT MEFs were infected with C. muridarum as in Fig. 3A and B. (B) Quantification of LAMP1-positive chlamydial inclusions revealed insignificant colocalization rates. Percentage of colocalization depicted. Error bars 6SD, n = 3. (C) No maturation of bacterial Control of Ctr via Irga6 inclusions into autophagosomes. WT, Irga62/2 and Atg52/2 MEFs were first transfected for 24 h with the autophagosome membrane marker GFP-LC3 and then exposed to 100 U/ml IFNc for an additional 24 h. Next, cells were infected with C. muridarum (MOI 5) for 8 h. IFNc-untreated control cells were similarly infected 48 h post-transfection. LC3 (green) bacterial inclusions (red) with very low processing for the LC3 (D) Quantification of GFP-LC3-positive C. muridarum inclusions demonstrate insignificant colocalization rates among different treatments. For quantification of both LAMP1 and GFP-LC3 +ve inclusions, around 300 inclusions were examined. Colocalization expressed as a mean percentage: number of GFP-LC3 +ve or LAMP1 +ve inclusions, respectively / total number of inclusions 6100. Error bars 6SD, n = 3. Scale bar represents 5 mm Found at: doi:10.1371/journal.pone.0004588.s002 (1.37 MB TIF) Figure S3 Irgd, Irgm2, Irgm3, Irgb6 and Irgm1 show a similar interaction pattern with C. trachomatis early inclusions in Irga6- knockout MEFs under stimulation with IFNc. Irga6-deficient cells were exposed to the cytokine, infected with the pathogen, and stained for Chlamydia and IRGs exactly as described in the legend to Fig. 2. Untreated knockout cells were infected and stained in parallel for comparison reasons. (A) Confocal micrographs showing the double labelling of cells with antibodies against the pathogen and different IRGs. (B) Quantification of IRG colocalization with C. trachomatis inclusions. Around 300 bacterial inclusions examined. Colocalization expressed as a mean percent- age: number of IRG +ve inclusions / total number of inclusions 6100. Error bars 6SD, n = 3. Scale bar represents 5 mm. Found at: doi:10.1371/journal.pone.0004588.s003 (0.67 MB TIF) Figure S4 Irga6 does not play a role in the regulation of the host autophagic machinery during C. muridarum infection. (A–C) Anti- LC3 immunoblot analysis of total lysates from uninfected WT, Atg52/2 and Irga62/2 MEFs or from cultures infected for indicated time points. Some uninfected cell cultures were exposed to 100 nM Rapa for 3 h or to 100 U/ml IFNc for 32 h. Other monolayers were pretreated with IFNc for 24 h prior to infection and then infected in the presence of chemicals. 1–7 indicate the different treatments. Autophagy induction is reflected by the increased cellular level of LC3 and formation of autophagosome- associated LC3-II. Host b-actin was used to control equal loading of proteins. After infection with C. muridarum, LC3 II level increases but to a less extent than C. trachomatis (Fig. 7) in IFNc treated (A) WT cells. LC3 levels do not increase in IFNc treated (C) Irga62/ 2 cells. (B) Defective autophagy in Atg52/2 MEFs was observed, indicated by absence of LC3 processing. Found at: doi:10.1371/journal.pone.0004588.s004 (0.39 MB TIF) Acknowledgments The authors would like to thank Stefan H.E. Kaufmann for providing access to the Irga62/2 knockout mice, generated in the Department of Immunology, Drs. Mischo Kusar and Markus Koch for preparing fibroblasts from these mice, and Drs. Phoebe Harjes and Lesley Ogilvie for expert assistance in preparing the manuscript. Author Contributions Conceived and designed the experiments: MAAZ HMAY PRB TFM. Performed the experiments: MAAZ HMAY. Analyzed the data: MAAZ HMAY. Contributed reagents/materials/analysis tools: MAAZ HMAY PRB JZ. Wrote the paper: MAAZ HMAY. PLoS ONE | www.plosone.org 12 February 2009 | Volume 4 | Issue 2 | e4588