Localization analysis of IQGAP1, CHMP2A, and ATG9A and effects of their depletion on autophagosome closure. (A) Huh7WT cells were transiently co-transfected with pDest-3xFLAG-ATG9A and pDest-GFP-CHMP2A and stained for endogenous IQGAP1. Cells were starved in EBSS for 90 min. The selected area is shown within the white square, and the inset is depicted in the merged image. The region of interest (ROI) is marked with a diagonal dashed white line. (B) Profile intensity (dashed diagonal in A inset) for multiple fluorescence channels. (C) Representative images (HCM), one of 80 fields/well in reference to Fig. 3 E. Huh7^WT and Huh7^ATG9AKO cells stably expressing HT-LC3B complemented with pDest-3xFLAG-ATG9A^WT or pDest-3xFLAG-ATG9A^M33 (lipid scramblase mutant). (D) Immunoblot analysis of ATG9A, CHMP2A and IQAGP1 knockdown in Huh7 HT-LC3B cells. (E) Quantification, MIL/MPL HCM closure assay. HeLa HT-LC3B (stable cells) treated with siRNAs for CHMP2A (squares), IQGAP1 (triangles), ATG9A (diamonds), or control (Scr; circles). Autophagy was induced in EBSS for 90 min ± BafA1 (100 nM). Cells were sequentially incubated with MIL to stain unclosed structures and MPL to stain HT-LC3B-II available in closed membrane. (i–iii) MPL⁺, (red) puncta/cell, (ii) MIL⁺ (green) puncta/cell; (iii) MIL/MPL (gray) ratios of puncta per cell in I and ii. (F) HCM images represent examples (1 of 80 fields/well; >500 primary objects (cells)/well; 4 wells per sample/plate) of MPL⁺ (red masks; closed) and MIL⁺ (green masks; unclosed) quantified in E. Data means, ± SD, n = 5 (biologically independent experiments); two-way ANOVA followed by Tukey’s multiple comparison test Scale bars, 10 μm. Source data are available for this figure: SourceData FS2.