ATG9A colocalizes with CHMP2A and supports sequential stages in autophagosome biogenesis. (A) Confocal microscopy imaging of Huh7^WT cells transiently transfected with pDest-3xFLAG-ATG9A and GFP-CHMP2A and stained for endogenous LC3B. Cells were starved in EBSS for 90 min. White square, enlarged area in the inset (merged image; dashed diagonal line - section). (B) Profile intensity (dashed diagonal in A inset) for multiple fluorescence channels. (C) Immunoblot analysis of ATG9A KO in Huh7 cells and MIL/MPL HCM closure assay in Huh7^WT (circles) and Huh7^ATG9AKO (squares) cells stably expressing HT-LC3B; starvation-induced autophagy (EBSS, 90 min) ± 100 nM BafA1. (i–iii) Quantifications: (i) MPL⁺ HT-LC3B (red symbols); (ii) MIL⁺ HT-LC3B (green symbols); (iii) MIL/MPL ratios (puncta/cell; gray symbols). (D) HCM images: red masks, MPL⁺ profiles, green masks, MIL⁺ profiles. Quantification: >500 (cells)/well with 80 fields/well; 6 wells per sample/plate. Data, means ± SD, n = 5 (biologically independent experiments); one-way ANOVA followed by Tukey’s multiple comparison test. (E) Complementation analysis of Huh7^ATG9AKO HT-LC3B cells transfected with pDest-3xFLAG, pDest-3xFLAG-ATG9A^WT or pDest-3xFLAG-ATG9A^M33 mutant. Source data are available for this figure: SourceData F3.