Figure 2.

Arf6 regulates phagophore formation. (A) HeLa cells transiently expressing LC3-CFP, Atg16L1-Flag, and either GFP, Arf6-GFP, or Arf6 Q67L-GFP for 20 h were fixed and subjected to immunofluorescence with an anti-Flag antibody. Confocal images of LC3-CFP, Atg16L1-Flag, and either GFP, Arf6-GFP, or Arf6 Q67L-GFP are shown. Higher magnifications of the colocalizations are shown in the insets. The colocalization (Pearson’s coefficient) between Atg16L1-Flag vesicles and either GFP, Arf6-GFP, or Arf6 Q67L-GFP is shown. For colocalization, the data are means ± SD. n = 20 cells. (B) HeLa cells transiently expressing Arf6-HA, Arf6 Q67L-HA, or an empty vector and Atg4B C74A–mStrawberry as indicated for 20 h were cultured in basal conditions or amino acid and serum starvation medium for 1 h. Cells were fixed and subjected to immunofluorescence with anti-Atg12 and anti-HA antibodies. Confocal images of Atg12 (green), Arf6, and Atg4B C74A–mStrawberry (red) are shown. The data represent the means ± SD of the number of Atg12 vesicles per cell obtained from three independent experiments in which ≥200 cells were analyzed. Please note that individual channels of the pictures are shown in Fig. S1 B. Arrows indicate Atg12 vesicles, which are larger and brighter than the background-staining speckles. (C) HeLa cells transfected with two rounds of control or Arf6 siRNA for 5 d were fixed and subjected to automatic counting of Atg16L1-GFP or endogenous Atg12 vesicles. Representative confocal pictures are shown. The data represent the means ± SD of the number of Atg16L1-GFP vesicles or Atg12 vesicles per cell obtained from three independent experiments in which ≥200 cells were analyzed. kd, knockdown. Bars, 5 µm.