Figure 7. VAMP3 participates in the regulation of LC3-positive vacuole morphology. (A) BMDMs transiently expressing an EGFP-VAMP3 chimera were infected with Y. pseudotuberculosis for 30 min and then processed for CLSM. Bacteria (blue) are contained within EGFP-VAMP3-positive and LC3-AlexaFluor^® 555-negative vacuoles. Scale bar: 5 µm and insert magnification 4×. A fluorescence scan along the white line on the insert in the merged panel is shown in (B). (C) A pie chart displaying the distribution of bacteria as a function of EGFP-VAMP3 and LC3-AlexaFluor^® 555 labeling. In each experiment, at least 30 infected cells were quantified in a double-blind analysis. Values are quoted as the mean ± SEM from at least 3 independent experiments. (D) The panel shows a bacterium inside a double-membrane vacuole (as visualized by TEM) in a HeLa cell treated with VAMP3 siRNA (siVAMP3). HeLa cells were treated with VAMP3 siRNA and siGLO-GREEN siRNA, sorted according to the GFP signal, again treated with VAMP3 siRNA or siCTRL, infected with Y. pseudotuberculosis for 4 h, and then processed for TEM. Arrowheads show double-membrane vacuoles. (E) Quantification of YCVs with single or double membrane after 4 h p.i in HeLa cells treated with control siRNA (siCTRL), smart pool VAMP3 siRNA (siVAMP3 (SP)). For each experiment, 45 cells were analyzed. The mean of 3 independent experiments is indicated and the error bars correspond to the SEM (F) BMDMs transiently expressing EGFP-VAMP3 were infected with Y. pseudotuberculosis for 3 h and processed for TEM. Arrowheads show Y. pseudotuberculosis contained in a single-membrane vacuole. (G) TEM quantification of single- or double-membrane YCVs for 38 BMDMs transiently expressing EGFP-VAMP3 or EGFP. (H) HeLa cells transiently expressing EGFP (left) or EGFP-VAMP3 (right) were treated with rapamycin for 7 h and then processed for TEM. White arrowheads show double-membrane vacuoles. The complete data set is shown in Figure S3G. (I) Quantification of double-membrane vacuoles in HeLa cells transiently expressing EGFP or EGFP-VAMP3 and treated (or not) with rapamycin. In each experiment, at least 35 infected cells were quantified in a double-blind analysis.