Fig. 7: ATG2A-ANKFY1 mediate lipid transfer from endosomes to phagophores.

a U₂OS cells were transfected with non-targeting control siRNA or indicated siRNA and then cultured in regular DMEM. 48 h after transfection, cells were treated with 200 nM Torin 1 for 3 h, fixed and stained with DAPI and antibodies against WIPI2 and LC3. Cells were imaged under confocal microscope. Scale bars, 10 μm. b Quantification of the results in a. Data are mean ± SEM. n = 22 images containing 176 cells (siNC), 12 images containing 136 cells (siANKFY1), 18 images containing 246 cells (siATG2A/B), or 26 images containing 356 cells (siUVRAG) from two independent experiments. P-values are listed. c Schematic diagram of lipid transfer assay. Donor liposomes (61% DOPC, 30% POPE, 2% NBD-PE, 2% Rhod-PE, 5% PI3P) and acceptor liposomes (65% DOPC, 30% POPE, 5% PI3P) were mixed with ATG2A, ANKFY1 and WIPI4 as indicated. d FRET-based lipid transfer assay as in c. The lipid transfer was measured by the increase of NBD fluorescence intensity. e Quantification of results in d. Bars represent averages of the NBD signal observed after 7200 s in three independent experiments. Error bars represent standard deviations. Data are mean ± SD. **P ≤ 0.01; ns, not significant.