Pulse-chase analysis of Halo-ATG9 localization and degradation. (A) Representative micrographs of U2OS and Halo-ATG9A knockout (KO) cells pulse-labeled with JFX650 HaloTag ligand at 0 and 24 h after labeling to demonstrate the level of background fluorescence in the absence of a Halo-ATG9A. Left panel: fluorescence signal; right panel: bright field. (B) Representative micrographs of JFX650 labeled Halo-ATG9A at 0 h (top) and 24 h (bottom) after labeling in the endogenously edited cell line (left) or in the stable tetracycline-inducible Halo-ATG9A add-back in ATG9A knock-out cells (right two panels). ATG9A knock-out cells (same images as in A) were included as a comparison to assess non-specific background signal. (C) Fluorescence gel showing time-dependent cleavage of Halo-ATG9A in cells exposed to a single doxycycline pulse (P) or continuously grown in the presence of doxycycline (C), with progressive accumulation of a lower (∼34 kD) fluorescent band corresponding to the HaloTag protein. (D) Representative micrographs showing the overlap between the endoplasmic reticulum (marked with GFP-SEC61) and RFP-ATG9A. (E) Representative micrographs showing the overlap between Halo-ATG9A and transiently expressed RFP-ATG9A. Source data are available for this figure: SourceData FS5.