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

Figure S2.

Figure S2

VAMP7, Syntaxin 7, Syntaxin 8, and Vti1b Regulate Autophagic Activity, Related to Figure 2

(A) HeLa cells transfected with two rounds of control, VAMP7, syntaxin 7 (Stx7), syntaxin 8 (Stx8), Vti1b, or Atg16L1 siRNA for 5 days were lysed and subjected to western blotting with the indicated antibodies. For Atg16L1 knockdown, the cells were starved when indicated using HBBS medium treatment for 8 hr and treated during the last 4 hr with bafilomycin A1 (Baf A1) or DMSO (vehicle). Quantification of LC3-II/actin ratio is shown for Atg16L1 knockdown. The data represent the mean ± SD of the percentage of LC3-II/actin ratio obtained from three independent experiments.

(B–D) HeLa cells transfected with two rounds of control, Stx7, Stx8, or Vti1b siRNA for 5 days were treated during the last 16 hr with bafilomycin A1 (Baf A1) or DMSO (vehicle). Cells were lysed and subjected to western blotting with the indicated antibodies. Quantification of LC3-II/actin ratio is shown. The data represent the mean ± SD of the percentage of LC3-II/actin ratio obtained from three independent experiments.

(E) HeLa cells stably expressing LC3-GFP-mRFP were treated as indicated during the last 16 hr with bafilomycin A1 (Baf A1), fixed and subjected to a Cellomics ArrayScan VTI HCS Reader analysis. Quantification of the size of autophagic vesicles is shown. The data represent the mean ± SEM of the size of autophagic vesicles (A.U; arbitrary unit) of 1 experiment representative of three independent experiments performed where minimums of 2000 cells were automatically analyzed.

(F) HeLa cells stably expressing LC3-GFP-mRFP were transfected with two rounds of control or VAMP7 siRNA for 5 days and treated during the last 16 hr with bafilomycin A1 (Baf A1) or DMSO. Cells were starved for 4 hr, fixed, and subjected to automatic counting of LC3 vesicles. Quantification of total number of vesicles/cell, autophagosomes/cell (AV/cell) and autolysosomes/cell (AL/cell) is shown. At least 2000 cells were counted per experiment and the values are mean ± SEM of one representative experiment of three independent experiments performed.

(G) HeLa cells transfected with two rounds of control, VAMP7, Stx7, Stx8, Sec22B, or Vti1b siRNA for 5 days were subjected to Alexa 488-labeled transferrin internalisation assay and the amount of fluorescent-transferrin internalised under different knockdown conditions was measured by FACS and presented in the graph. NS—not significant.

(H) HeLa cells transfected with two rounds of control, VAMP7 or Stx7 siRNA for 5 days were lysed and subjected to western blotting with the indicated antibodies. Quantification of p62/actin ratio is shown. The data represent the mean ± SD of the percentage of p62/actin ratio obtained from three independent experiments.

(I) HeLa cells transfected with two rounds of control, Stx7, Stx8, or Vti1b siRNA for 5 days were fixed and subjected to immunolabeling for endogenous LC3 and Atg16L1. At least 50 cells were counted per experiment and the values are mean ± SD of the number of Atg16L1 vesicles per cell obtained from two independent experiments. The 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 obtained from two independent experiments. Scale bar, 5 μm.

(J) DFCP1-GFP cells transfected with two rounds of control or VAMP7 siRNA for 5 days were fixed and subjected to confocal analysis for DFCP1 dots counting. At least 50 cells were counted per experiment and the data are mean ± SD of the number of DFCP1 dots per cell (in percentage, relative to control) obtained from two independent experiments.