Figure 1(A) HeLa cells stably expressing GFP‐LC3_B (left panel) or GFP‐GATE‐16 (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.(B) Quantification of relative p62 and GFP‐GATE‐16 level (upper panel) and degradation (lower panel) was performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.(C) HeLa cells stably expressing GFP‐GATE‐16 were transfected with non‐targeting siRNA or a pool of LC3 siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.Figure 2(A) HeLa cells stably expressing GFP‐GATE‐16 (left panel) or GFP‐LC3_B (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE‐16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.(B) Quantification of relative p62 and GFP‐LC3_B level (upper panel) and degradation (lower panel) were performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.(C) HeLa cells stably expressing GFP‐LC3_B were transfected with non‐targeting siRNA or a pool of GABARAP siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.Figure 3(A) Bulk protein degradation was tested in HeLa cells that were transfected with non‐targeting siRNA (control siRNA), a pool of GABARAP/GATE‐16 siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either αMEM (control) or EBSS (starvation) medium, in the presence or absence of 10 mM of 3‐MA, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations.(B) HeLa cells stably expressing GFP‐LC3_B (left panel) or GFP‐GATE‐16 (right panel) were transfected with a pool of GABARAP/GATE‐16 siRNAs or a pool of LC3, respectively, and with non‐targeting siRNA. Seventy‐two hours after transfection, the cells were collected after 5 h of starvation and the relative level of GFP‐LC3_B or GFP‐GATE‐16 was measured using flow cytometry. Values represent the mean±s.d. of three experiments.Figure 4(A) HeLa cells stably expressing YFP‐Atg5 were transfected with non‐targeting siRNA (control siRNA), a pool of LC3 siRNAs, or a pool of GABARAP/GATE‐16 siRNAs using DharmaFect reagent. Seventy‐two hours after transfection, the cells were incubated for 2 h in EBSS medium, fixed, and immunostained with anti‐Atg16 antibodies. Quantification of Atg5 and Atg16‐labelled puncta structures from three independent experiments is presented on the right panel. Scale bar: 20 μm.(B) HeLa cells stably expressing YFP‐Atg5 were treated as in (A) and monitored by live microscopy. Quantification of the lifespan of Atg5‐labelled puncta structures is presented. Mean±s.d. of three independent experiments is presented. ^*P0.05, ^**P0.001.(C) YFP‐Atg5 cells were transfected with siRNA pool and starved as in (A). Cryo sections of fixed cells were immunolabelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of the Atg5‐labelled structures size is presented at the right panel. ^*P0.05, ^**P0.001.Figure 5(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3_B or GFP‐GATE‐16 were starved for 2 h in EBSS medium fixed and immunostained with anti‐Atg16 antibodies. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Arrowheads represent Atg‐16‐labelled puncta. Scale bar: 20 μm.(B) HeLa cells stably expressing GFP‐LC3_B (left panel) or GFP‐GATE‐16 (middle panel) were starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immunolabelled as in ( Figure 4B). Quantification of the size of GFP‐labelled structures (right panel) from three independent experiments is presented. ^*P0.05, ^**P0.001.Figure 6(A, B) Control HeLa cells and HeLa cells stably expressing GFP‐LC3_B (A) or GFP‐GATE‐16 (B) were transfected with the reciprocal siRNA pools using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS, subjected to immunostaining with anti‐Atg16 after fixation, and analysed by confocal microscopy. Quantification of cells containing Atg16 structures larger than 2 μm (A) or Atg16‐labelled puncta structures (B) from three independent experiments in comparison to control and to G to A mutants (D) is presented at the lower panel. Scale bar: 20 μm.(C) HeLa cells stably expressing silent GFP‐LC3_B or GFP‐GATE‐16 were transfected with their siRNA pools using DharmaFect reagent. After 72‐h interval, the cells were incubated for 2 h in EBSS medium and subjected to immunostaining with anti‐Atg16 and anti‐p62 antibodies followed by fixation. The cells were analysed by confocal microscopy. Arrows represent cells, which do not express GFP proteins whereas arrowheads represent cells, which express GFP‐proteins. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Scale bar: 20 μm.(D) HeLa cells stably expressing GFP‐LC3_B^G120A or GFP‐GATE‐16^G116Awere transfected with either pool of GABARAP/GATE‐16 siRNAs or LC3 siRNAs, respectively, and a non‐targeting siRNA. The cells were starved for 2 h, fixed, and immunostained with anti‐Atg16 and anti‐p62 antibodies. Quantification is presented in (A, B) at the lower panels. Scale bar: 20 μm.(E) HeLa cells stably expressing GFP‐LC3_B (left panel) or GFP‐GATE‐16 (right panel) were transfected with the reciprocal siRNA pool or with non‐targeting siRNA and starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immuno‐labelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of cells containing Atg16 structures larger than 2 μm (left panel) or the size of Atg16‐labelled structures (right panel) is presented.Figure 7(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3_B or GFP‐GATE‐16 were transfected with HsAtg4A^C77A‐Myc‐His6. After 48 h interval, the cells were incubated for 2 h in EBSS, and subjected to immunostaining with anti‐Atg16 and anti‐myc followed by confocal microscopy analysis. Quantification of cells containing Atg16 structures larger than 2 μm from three independent experiments is presented at the right panel. Scale bar: 20 μm.(B) HeLa cells were transfected with HsAtg4A^C77A‐Myc‐His6 or empty vector (mock). After a 48 h interval, the cells were incubated for 2 h in EBSS, fixed, and their ultrathin sections were analysed by TEM as described in 'Materials and methods'. ^*P0.05.