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. 2014 Jul 10;10(9):1588–1602. doi: 10.4161/auto.29411

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Figure 5. VAMP7 has a role in the recruitment of LC3 to YCVs-LAP. (A) HeLa cells transiently expressing a GFP-LC3 chimera and treated with control siRNA (siCTRL) were infected with Y. pseudotuberculosis for 4 h and then processed for CLSM. VAMP7 proteins were immunostained and visualized with a secondary antibody coupled to AlexaFluor® 555. Bacteria (blue) are contained in GFP-LC3- (green) and VAMP7-AlexaFluor® 555- (red) positive vacuoles. Scale bar: 5 µm. Insert magnification: 4×. A fluorescence scan along the white line in the insert is shown in the right panel (B). (C) HeLa cells transiently expressing a GFP-LC3 chimera and treated with VAMP7 siRNA (siVAMP7) were infected with Y. pseudotuberculosis for 4 h and then processed for CLSM. VAMP7 proteins were immunostained and visualized with a secondary antibody coupled to AlexaFluor® 555 (red). Bacteria (blue) are contained in GFP-LC3-positive (green) and VAMP7-AlexaFluor® 555-negative vacuoles. Scale bar: 5 µm. Insert magnification: 5×. A fluorescence scan along the white line in the merged insert is shown on the right (D). (E) The percentages of YCVs displaying a GFP-LC3 labeling were quantified in HeLa cells treated or not with VAMP7 siRNA. In each experiment, at least 20 cells infected with Y. pseudotuberculosis during 4 h were quantified. (F) HeLa cells transfected with control or VAMP7 siRNA were analyzed for LC3 by immunoblotting. The cells were treated with rapamycin or BafA1 as indicated and DMSO was used as a solvent control. Protein loading was checked against the α-tubulin (TUBA) content. The complete data set is shown in Figure S2A. (G) The LC3-II/TUBULIN ratio for the HeLa siCTRL condition as a function of cell treatment or infection. The data correspond to the mean of 3 independent experiments and the error bars correspond to the SEM (H) VAMP7’s role in Yersinia-activated autophagy is shown by the [(LC3-II/TUBULIN)infected]/[(LC3-II/TUBULIN) uninfected] ratio for HeLa cells treated with control siRNA (siCTRL) or siRNA against VAMP7 (siVAMP7) and as a function of drug treatments. The data correspond to the mean of 3 independent experiments and the error bars correspond to the SEM.