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. 2018 Feb 9;9:599. doi: 10.1038/s41467-018-02823-x

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — BRUCE is required for starvation-induced degradation of ATG8 family proteins. a Normalized mCherry and GFP signals after starvation compared to basal condition (FM) analyzed by flow cytometry in Bruce^+/+ and Bruce^−/− MEFs stably expressing mCherry-EGFP-LC3B. Data are presented as mean±SD from three biological replicates (****p < 0.0001). Representative data are shown from four independent experiments. b Confocal microscopy images of Bruce^+/+ and Bruce^−/− MEFs stably expressing mCherry-EGFP-LC3B in different conditions. FM, fully supplemented medium; Starv, starvation medium for 4 h. Scale bars, 20 µm. c Quantification of mCherry-GFP double positive spots in Bruce^+/+ and Bruce^−/− MEFs in regular and starvation conditions (as in b). Data are presented as dot plots with mean±SD. n = 20 cells. p-values are indicated and analyzed by t-test. d Protein levels of autophagy markers in Bruce^+/+ and Bruce^−/− MEFs under basal (FM) and 2 h-starved (Starv) conditions with or without Bafilomycin A1. e A schematic of the inserted gene trap cassette in Bruce^−/− haploid mouse embryonic stem cells (mESCs). f Protein levels of autophagy markers in Bruce^+/+ and Bruce^−/− clone #1 haploid mESCs under basal (FM) and 2 h-starved (Starv) conditions with or without Bafilomycin A1. BRUCE expression was recovered by Cre recombinase in Bruce^−/− mESCs and compared with mock infected mESCs