Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy ATG16L1−/− cells reconstituted with ATG16L1WT or ATG16L1LE.

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Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy ATG16L1−/− cells reconstituted with ATG16L1WT or ATG16L1LE were cultured in full growth media prior to immunofluorescence analyses using antibodies against WIPI2 (green) or ATG16L1 (red). Scale bar: 9 μm. Cells as in (A) with the addition of 3′MA treatment for 30 min. Scale bar: 9 μm. Quantification of percentage of cells positive for ATG16L1 puncta in (A) and (B). Quantifications depict means and error bars (SEM) from at least three independent experiments. ns P > 0.05, ***P ≤ 0.001 (pairwise unpaired Student's t‐test). Ferroptosis assay in ATG16L1−/− cells stably expressing F‐S‐tagged ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 h. Representative PI staining and phase contrast images are shown (relevant to Fig G). Scale bar: 30 μm. Western blot analyses of ATG16L1−/− cells stably expressing F‐S‐tagged ATG16L1WT, ATG16L1LD or ATG16L1LE using the indicated antibodies (relevant to D and Fig G). IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 38, Issue: 9, First published: 01 April 2019, DOI: (10.15252/embj.2018100554)