Loss of GABARAP impairs autophagy induction and alters Golgi morphology. (A) AMO1 and H929 WT and GABARAP^KO were subjected to proteomic analysis by multiplexed proteomics with mass spectrometry. Shown in panel A is the gene set enrichment analysis (GSEA) gene ontology cellular components (GOCC) that were significantly negatively enriched after GABARAP KO. (FDR <1% for AMO1 and FDR <25% for H929). (B-C) Analysis of autophagy in AMO1 WT and GABARAP^KO cells by TEM. (B) Representative TEM images depicting Golgi morphology (A = double-layered vesicles); scale bars, 500 nm. (C) Histograms showing the number of double-layered vesicles as determined in a total of 30 images for AMO1 WT and 30 images for AMO1 GABARAP^KO cells. (D) AMO1 WT, GABARAP^KO, and GABARAP^KO in which GABARAP was re-expressed (GABARAP^KO + add-back) were left untreated or treated with BTZ (5 nM; 16 hours). Immunoblot of GABARAP and LC3A/B is shown. β-Actin was used as a loading control. (E) Representative confocal images of Golgi apparatus stained with GM-130 antibody (green) in AMO1 WT, GABARAP^KO, and GABARAP^KO treated with rapamycin (50 nM; 24 hours). DAPI was used to label nuclei. This merged figure is also reported as supplemental Figure 4L together with the ones of the single channels; scale bars, 20 μm. (F) Box plot showing the Golgi area (μm²) in the different conditions as determined in a total of 119 cells per condition for AMO1, 60 cells per condition for H929, and 60 cells per condition for U266. (G) Representative TEM images depicting Golgi morphology in AMO1 WT, GABARAP^KO, and GABARAP^KO treated with rapamycin (50 nM; 24 hours) (C = compact; D = dispersed; and S = swollen); scale bars, 500 nm. (H) Histogram showing the percentage of compact, swollen, and dispersed Golgi in each condition. Specifically, 61 Golgi were visible in 29 TEM images taken in AMO1 WT; 37 Golgi in 30 TEM images taken in AMO1 GABARAP^KO; and 47 Golgi in 29 TEM images taken in AMO1 GABARAP^KO treated with rapamycin. For panel C, ∗∗P < .01 based on the unpaired Student t test; for panel F, ∗∗∗∗P < .0001 Kruskal-Wallis test. RAPA, rapamycin.