In vitro assay for autophagosome closure. (A) SolVit (sealing of organellar limiting membranes in vitro) assay schematic: in vitro complementation by mixing postnuclear supernatants (PNS) from ATG9A^KO HT-LC3B cells (Acceptor) with PNS from ATG9A^WT or ATG9A^KO cells (Donor), ±ATP, incubated for 1 h. PNS were from cells treated with 20 μM CCCP for 6 h. Reaction products were stained with MIL and MPL sequentially and immobilized in mounting media on the bottom of 96-well plates followed by HCM quantification. (B i–iii) MPL⁺ profiles (red); (ii) MIL⁺ profiles (green). (iii) MIL/MPL ratios (gray). Each HCM experimental point: 1,000 valid primary objects/cells per well, 5 wells/sample. Data, means ± SD, n = 3 (biologically independent experiments); one-way ANOVA followed by Tukey’s multiple comparison test. (C) Examples of HCM images from SolVit assay. Red profiles, MPL⁺ closed LCB⁺ membranes; Green profiles, MIL⁺ unclosed LC3B⁺ membranes. Scale bars, 3 μm. Source data are available for this figure: SourceData F4.