Figure 2.Yersinia pseudotuberculosis is contained within single-membrane, nonacidic, LC3-positive vacuoles. (A) HeLa cells stably expressing mRFP-GFP-LC3 were infected with Y. pseudotuberculosis for 4 h and then processed for CLEM. Bacteria were visualized after staining with DAPI. The upper panel shows a single HeLa cell observed by CLSM and the middle panel shows the corresponding TEM image. The lower left images show bacteria inside an mRFP-GFP-LC3 vacuole (insert magnification: 8×). The lower right image shows bacteria contained inside a single-membrane vacuole (arrowheads). (B) Bacteria dividing inside a single-membrane vacuole, as visualized by TEM. HeLa cells stably expressing mRFP-GFP-LC3 were infected with Y. pseudotuberculosis for 4 h and then processed for electron microscopy. (C) A quantitative TEM analysis of YCVs with single or double membranes. Values are quoted as the mean ± SEM from 3 independent experiments in which at least 20 HeLa cells stably expressing mRFP-GFP-LC3 were analyzed.