Fig 2: dSTORM imaging of ERGIC.Parental HEK293 cells (a,b,d) or HEK293 cells stably expressing GFP-DFCP1 (c) were starved, immunolabelled for ERGIC53 and ATG9 (a), SEC23 (b), GFP(c) or ATG16 (d) and imaged by dSTORM. Representative conventional images and super-resolution magnifications are shown. Scale bars in wide-field images, 5 μm. Scale bars in super-resolution images, 0.5 μm.

Fig 3: TFG downregulation affects ULK1‐ATG9 co‐localizationHEK293 cells stably expressing GFP‐ATG13 were interfered or not with TFG siRNA (siTFG) and starved for 1 h. Cells were fixed and immunolabelled with ATG9 (red) antibody. Co‐localization analysis was performed by Jacop plugin. Values of Mander’s coefficient for ULK1 are reported as percentage and represented as mean ± SEM. Significance was assigned by two‐way ANOVA followed by Tukey’s multiple comparison test. **P < 0.01 and ***P < 0.001. Scale bar 5 μm. N, nucleus (n = 3 independent experiments, n ≥ 20 fields analysed).

Fig 4: ATG9 promotes the formation of ATG13 puncta.(a) HEK293 cells were either fed or starved for 1 h, immunolabelled for ATG13 and ATG9 and imaged by wide-field microscopy. Arrowheads in inserts point at ATG13 particles associating with ATG9. Bar corresponds to 10 μm. (b) Values are ATG13 particles in c,d associating with ATG9 particles in the first two frames from their emergence. From analysis of 75 montages. (c,d) Wide-field live-cell imaging of starved HEK293 cells stably expressing GFP-ATG13 and mRFP-ATG9. Representative montages of ATG13 particles forming in association with ATG9 (c) or not (d) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in b. (e) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and mRFP-ATG9, and transiently CFP-ER. Representative montage of ATG13 particle forming on a tubular extension of ER previously hosting an ATG9 vesicle is shown. Arrowheads point at the ATG13 particle in the first two frames from its emergence and at the associating extension of ER and ATG9 vesicle. (f,g) HEK293 cells were transfected with non-targeted (siNT) or ATG9 (siATG9) siRNA, starved for 1 h, immunolabelled for ATG13 or WIPI2 and imaged by confocal laser scanning microscopy. Values are means±s.e.m. puncta of ATG13 (f) or WIPI2 (g) per cell, for at least five different fields with 15–30 cells each. Significance levels were determined with unpaired t-tests. Bar in c–e corresponds to 300 nm. **P=0.01%; ***P=0.001%.

Fig 5: Autophagosomes associate with ATG9 and ERGIC membrane compartments.(a,b) HEK293 cells were starved in the presence or absence of VPS34 inhibitor for 1 h, immunolabelled for ATG13 and ATG9 (a) or FIP200 and ERGIC53 (b), and imaged by dSTORM. (c,d) HEK293 cells stably expressing GFP-ATG13 were starved in the presence or absence of Vps34 inhibitor for 1 h, immunolabelled for ATG13 and ATG9 (c) or SEC23 (d), and imaged by dSTORM. Conventional images and super-resolution magnifications are shown. Scale bars in wide-field images: 5 μm. Scale bars in super-resolution images, 0.5 μm.