IQGAP1 contributes to autophagosome closure via CHMP2A. (A) HCM Images (example) of the effects of IQGAP1 knockdown in Huh7-HT-LC3 cells in Fig. 1 E, (masks: white, primary objects; green, MIL⁺ profiles; red, MPL⁺ profiles). (B) Immunoblot analysis of IQGAP1 knockdown with individual siRNAs, siRNA1 or siRNA2 in Huh7 HT-LC3B cells. (C) HCM example images corresponds to Fig. 1, F i–iii. (D) Upper panel, expression analysis by immunoblotting of the siRNA1 or siRNA2 resistant constructs pDest-3xFLAG-IQGAP1^Res1 and pDest-3xFLAG-IQGAP1^Res2 in Huh7 HT-LC3B cells. Lower panel, examples of HCM images of FLAG⁺ (gated) cells transfected with pDest-3xFLAG-IQGAP1^Res1 or pDest-3xFLAG-IQGAP1^Res2, corresponding to Fig. 1, F i–iii. (E) Immunoblot analysis of IQGAP1 knockdown in Huh7 HT-LC3B cells (top blot) and protease protection assay (bottom blot) of p62 and NDP52 in Huh7 HT-LC3B cell extracts (siRNAs: Scr, siRNA control; siIQGAP1, IQGAP1 siRNA) ± proteinase K with or without Triton X-100 treatment. Quantification of p62 and NDP52 levels (band intensities) in control and proteinase K-treated samples. Data are means ± SD, n = 3 (biologically independent experiments); one-way ANOVA followed by Tukey’s multiple comparison test. (F) Quantification by immunoblotting (band intensity) of CHMP2A in whole cell lysates in control cells and cells knocked down for IQGAP1 (corresponding to Fig. 2, D ii). (G) Quantification (i–iii) and example images (iv), MIL/MPL HCM assay in Huh7 HT-LC3B cells treated with control (siScr) or CHMP2A siRNA (siCHMP2A) and sequentially incubated with membrane-impermeable HT ligand (MIL) to stain HT-LC3B-II (cytosolic) and membrane-permeant HT ligand (MPL) to stain LC3B-II, sequestered within closed membrane. HCM images: MPL⁺ (red mask) and MIL⁺ (green mask) puncta. Huh7 HT-LC3B cells were incubated in EBSS to induce autophagy for 90 min ± BafA1 (100 nM). Scale bars, 10 μm. Source data are available for this figure: SourceData FS1.