ATG9 does not detectably accumulate at the site of autophagosome formation. (A) Example image showing the formation of GFP-P62 spot in the absence of any Halo-ATG9A accumulation. Scale bar = 1 μm. (B) Fluorescent gels showing Halo-ATG9A (JFX650, top) and SNAP-LC3B (JFX650, middle) labeling and Western blot showing probed with an LC3 antibody showing the exclusive expression of SNAP-LC3B in genome-edited cells. (C) Micrographs of cells expressing Halo-ATG9A (JFX650) and SNAP-LC3B (JF503; top, scale bar = 10 µm) and kymograph of a SNAP-LC3B spot showing no accumulation of Halo-ATG9A (one frame per second, frames 79–360). (D) Fluorescent gel of SNAP-LC3B (JFX650) after cell starvation and treatment with bafilomycin, demonstrating lipid conjugation of SNAP-LC3B (bottom band). (E) Fluorescence gel and Western blots demonstrating successful ATG9A gene knockout from cells expressing Halo-ATG2A. The ATG9A knock-out cells accumulate P62, indicating impaired autophagy. (F) Micrographs showing a decrease of Halo-ATG2A foci when ATG9A is knocked out (scale bar = 10 μm). Halo-ATG9A does not form detectable foci under EBSS starvation (right panel). (G) Histograms of Halo-ATG2A foci lifetime in parental and ATG9A knock-out cells (EBSS, 1 h). (H) Quantification of the number of foci formed per cell over the course of an hour by Halo-ATG2A in control and ATG9A knock-out cells. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis (*P < 0.05). Source data are available for this figure: SourceData F7.
Figure 7.

ATG9 does not detectably accumulate at the site of autophagosome formation. (A) Example image showing the formation of GFP-P62 spot in the absence of any Halo-ATG9A accumulation. Scale bar = 1 μm. (B) Fluorescent gels showing Halo-ATG9A (JFX650, top) and SNAP-LC3B (JFX650, middle) labeling and Western blot showing probed with an LC3 antibody showing the exclusive expression of SNAP-LC3B in genome-edited cells. (C) Micrographs of cells expressing Halo-ATG9A (JFX650) and SNAP-LC3B (JF503; top, scale bar = 10 µm) and kymograph of a SNAP-LC3B spot showing no accumulation of Halo-ATG9A (one frame per second, frames 79–360). (D) Fluorescent gel of SNAP-LC3B (JFX650) after cell starvation and treatment with bafilomycin, demonstrating lipid conjugation of SNAP-LC3B (bottom band). (E) Fluorescence gel and Western blots demonstrating successful ATG9A gene knockout from cells expressing Halo-ATG2A. The ATG9A knock-out cells accumulate P62, indicating impaired autophagy. (F) Micrographs showing a decrease of Halo-ATG2A foci when ATG9A is knocked out (scale bar = 10 μm). Halo-ATG9A does not form detectable foci under EBSS starvation (right panel). (G) Histograms of Halo-ATG2A foci lifetime in parental and ATG9A knock-out cells (EBSS, 1 h). (H) Quantification of the number of foci formed per cell over the course of an hour by Halo-ATG2A in control and ATG9A knock-out cells. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis (*P < 0.05). Source data are available for this figure: SourceData F7.

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