Disrupted ATG9A–IQGAP1 interaction impairs autophagosomal closure. (A–D) ^TMRHT release assay (see Fig. 2) in Huh7^WT or Huh7^ATG9AKO cells stably expressing HT-LC3B. Huh7^ATG9AKO transfected with pDest-3xFLAG, pDest-3xFLAG-ATG9A^WT, pDest-3xFLAG-ATG9A^CD1, or pDest-3xFLAG-ATG9A^CD2. Free ^TMRHT was measured by in-gel fluorescence (A and B) and immunoblotting (C and D). Cells were induced for autophagy in EBSS (90 min). Data are means ± SD, n = 3 (biologically independent experiments); one-way ANOVA followed by Tukey’s multiple comparison test. (E) MIL/MPL HCM assay in Huh7^ATG9AKO HT-LC3B cells transiently transfected with pDest-3xFLAG-ATG9A^WT (circles), pDest-3xFLAG-ATG9A^CD1 (squares), or pDest3xFLAG-ATG9A^CD2 (triangles). Gating on FLAG immunofluorescence was used to identify transfected cells. (i–iii) MPL⁺, closed HT-LC3B profiles (red symbols); (ii) MIL⁺, accessible HT-LC3B (green symbols); (iii) MIL/MPL ratios (gray symbols) of puncta/cell values in (i) and (ii). Cells were starved in EBSS for 90 min ± 100 nM BafA1, sequentially incubated with HT ligands MIL and MPL with immunostaining of FLAG incorporated into the protocol. Quantification: >500 cells/well; 4 wells per sample/plate, Data, means ± SD, n = 4 (biologically independent experiments); ANOVA followed by Tukey’s multiple comparison test. Source data are available for this figure: SourceData F7.