ATG9A knockout displays little to no lipidation. (A) Immunoblot showing the loss of ATG9A protein in ATG9A KO HEK293 cells. LC3B lipidation is blocked and p62 accumulates, suggesting no autophagic flux. Loaded protein: 10 µg. (B) Immunofluorescence of WT HEK293 cells compared to ATG9A KO HEK293 cells and ATG9A KO cells rescued with transient expression of FLAG-ATG9A. Rescue of the ATG9A KO shows restoration of bafilomycin A1-dependent accumulation of autophagosomes as in the WT. Maximum intensity projections of confocal images. Scale bars: 10 µm. (C) Immunoblots showing LC3B lipidation, p62 turnover, and bafilomycin A1-induced accumulation of lipidated LC3B is restored by addition of exogenous ATG9A in both transient (left) and stable (right) expression in ATG9A KO cells. Loaded protein: 10 µg. (D) Immunoblot showing the loss of FIP200 protein in FIP200 KO HEK293 cells. LC3B lipidation is blocked, suggesting no autophagic turnover. Loaded protein: 10 µg. (E) Immunoblot showing the loss of ATG3 protein in ATG3 KO HEK293 cells. Loss of LC3B lipidation suggests no autophagic turnover. Loaded protein: 15 µg.