Skip to main content
. 2011 Jul 22;146(2):303–317. doi: 10.1016/j.cell.2011.06.023

Figure S3.

Figure S3

Homotypic Fusion of Atg16L1 Vesicles Analysis, Related to Figure 3

(A) HeLa cells transfected for 20 hr with Atg16L1-GFP and LC3-mRFP were subjected to live cell imaging. Representative images from 5 min movies are shown. Note that two Atg16L1-GFP vesicles without LC3 fused together. Arrows show Atg16L1-GFP vesicles involved in fusion event. Scale bar, 5 μm.

(B) Time course of the in vitro fusion assay of Atg16L1 vesicles. Postnuclear supernatants (PNS) of cells expressing Atg16L1-GFP were mixed with PNS from cells expressing Atg16L1-Flag for 10 min, 30 min, or 50 min. The reaction were put on coverslips and analyzed by confocal microscopy after an immunostaining for Flag. The data represent the mean ± SD of the percentage of Atg16L1-GFP vesicles fused (colocalized) with Atg16L1-Flag. n = 30 vesicles for 0 min, n = 33 vesicles for 10 min, n = 79 vesicles for 30 min, n = 93 vesicles for 50 min obtained from two independent experiments.

(C) Immunostaining of endogenous LC3 in the in vitro fusion assay of Atg16L1 vesicles without ATP. After the in vitro fusion reaction without ATP, the Atg16L1 vesicles were stained for endogenous LC3 and analyzed by confocal microscopy. A representative confocal picture is shown where Atg16L1 vesicles do not contain LC3 (arrows) whereas one Atg16L1 vesicle does contain LC3 (arrowhead). The graph represents the percentage of Atg16L1 vesicles with LC3. The data are the mean ± SD n = 60 vesicles obtained from two independent experiments.

(D) Postnuclear supernatants (PNS) of cells expressing Atg16L1-GFP were mixed with PNS from cells expressing either Atg16L1-Flag for 1 hr, in absence of ATP or presence of NEM. The reaction was put on coverslips and analyzed by confocal microscopy. On the left, the data represent the average size ± SD of Atg16L1-GFP vesicles (arbitrary unit, A.U). n = 163 vesicles for untreated condition, n = 65 vesicles for NEM-treated condition, n = 159 vesicles for ATP-untreated condition obtained from two independent experiments. On the right, the data represent the average size ± SD of unfused Atg16L1-GFP vesicles with Atg16L1-Flag (only green) or fused Atg16L1-GFP vesicles with Atg16L1-Flag (yellow). n = 25 vesicles for unfused vesicles, n = 25 vesicles for fused vesicles.

See also Movie S1.