Figure 2.

VAMP7 Regulates Autophagic Activity

(A) HeLa cells transfected with control or VAMP7 siRNA for 5 days were treated during the last 16 hr with bafilomycin A1 (Baf A1) or DMSO. Means ± SD of the percentage of LC3-II/tubulin ratio from three independent experiments are shown. LE (light exposure of film), HE (longer exposure of film).

(B) HeLa cells transfected with control or VAMP7 siRNA for 5 days were starved and treated during the last 16 hr with bafilomycin A1 (Baf A1) or DMSO. Means ± SD of LC3-II/actin ratio from three independent experiments.

(C) HeLa cells stably expressing LC3-GFP-mRFP were transfected with control or VAMP7 siRNA for 5 days and treated during the last 16 hr with bafilomycin A1 (Baf A1) or DMSO. Cells were fixed and subjected to automated counting of LC3 vesicles. Representative confocal pictures are shown. Scale bar, 5 μm.

(D) Quantification of total number of vesicles/cell, autophagosomes/cell (AV/cell; LC3 GFP⁺/mRFP⁺), and autolysosomes/cell (AL/cell; LC3 GFP⁻/mRFP⁺) from (C) is shown. At least 2000 cells were counted per experiment; values are mean ± SEM of one representative experiment of three independent experiments performed.

(E) HeLa cells transfected with control, VAMP7, Ulk1, or Sec22B siRNA for 5 days were immunolabeled for endogenous LC3 (red) and Atg16L1 (green). Representative confocal pictures and quantification of Atg16L1 vesicles are shown. At least 50 cells were counted per experiment; values are mean ± SD of the number of Atg16L1 vesicles per cell obtained from two independent experiments. Colocalization efficiency between Atg16L1 and LC3 is also shown. At least 50 cells were counted per experiment and the data represent the percentage of Atg16L1 vesicles containing LC3 ± SD obtained from two independent experiments.

Scale bar, 5 μm. NS—not significant. See also Figure S2.