Control of Ctr via Irga6 Figure 5. Absence of Irga6 colocalization at C. trachomatis early inclusions in IFNc stimulated Atg52/2 MEFs. (A) and (B) IFNc treated and untreated cells were infected with C. trachomatis as in Figure 2. (A) Confocal microscopic analysis of infected stimulated Atg5-knockout cells shows similar staining patterns for Irgb6, Irgd, Irgm1, Irgm2 and Irgm3 as in stimulated infected WT MEFs (Figure 2). Irga6 does not localize to the inclusion in IFNc-stimulated and unstimulated Atg5-knockout cells. (B) Quantification of colocalization rates of IRGs with C. trachomatis inclusions. Around 300 bacterial inclusions were examined. Colocalization expressed as a mean percentage: for each treatment, number of IRG +ve inclusions / total number of inclusions 6100. Error bars 6SD, n = 3. Scale bar represents 4 mm. doi:10.1371/journal.pone.0004588.g005 membrane of nascent autophagosomes and correlates with the amount of LC3-positive autophagosomes [30]. In WT cells, LC3- II levels increased in response to IFNc stimulation (Figure 7A, lane 3). Notably, levels of IFNc-induced LC3-II were comparable to those induced by rapamycin (Rapa), a conventional inducer of autophagy (Figure 7A, lane 2). Infection with C. trachomatis for 4 h or 8 h did not influence LC3-II expression (Figure 7A, lanes 4 and 6, respectively). However, IFNc treatment of infected WT MEFs induced an upregulation of LC3-II (Figure 7A, lanes 5 and 7). In general, levels of LC3 protein were substantially lower in Irga62/ 2 cells (Figure 7C) as compared to WT MEFs (Figure 7A). Moreover, IFNc or Rapa treatment had a minimal stimulatory effect on LC3 expression and/or processing in Irga62/2 cells (Figure 7C, lanes 2, 3, 5 and 7). C. muridarum infection effectively suppressed an increase in LC3 levels upon IFNc treatment (Figure S4A). Similarly, no increase in LC3 levels upon IFNc treatment in C. muridarum infected Irga62/2 cells (Figure S4C) was observed. Atg52/2 cells did not process LC3 (Figure 7B and S4B), as expected for cells that are unable to form autophagosomes [31]. To confirm the importance of Irga6 in autophagy, we quantified the number of autophagosomes in the cytoplasm. In contrast to control untreated WT cells (Figure 7D), numbers of GFP-LC3- decorated structures per cell in infected and uninfected WT cells exposed to either IFNc or Rapa were increased. In agreement with our immunoblot analysis, IFNc or Rapa induction did not increase the amount of autophagosomes and we observed only low numbers of decorated vesicles in Irga62/2 cells (Figure 7E). These data further support a pivotal role for autophagy in C. trachomatis elimination and indicate a role for Irga6 as regulator of IFNc-induced autophagy. PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4588