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. 2020 Jan 31;11:645. doi: 10.1038/s41467-019-14164-4

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© The Author(s) 2020

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. 1 — a CMA-targeting motif in Dendra to generate the KFERQ-Dendra mouse lines. Right: CMA steps. b Representative immunoblot for Dendra in homogenates (Hom) and lysosomes (Lys) from the indicated tissues of fed (F) or 24 h starved (S) KFERQ-Dendra mice. All samples per experiment were run in the same membrane but for each protein different membranes were used. Similar results were obtained in three independent experiments. c Green fluorescence (from KFERQ-Dendra) and immunofluorescence (for LAMP1) of livers from 24 h starved KFERQ-Dendra mice. Bottom: Higher magnification of the dotted square. Arrows: examples of LAMP1-positive compartments with KFERQ-Dendra. Similar results were obtained in four independent experiments. d Immunofluorescence for Dendra (pseudocolor in green) in liver sections from KFERQ-Dendra mice 12 h after injection with PBS (None) or chloroquine. Blue: DAPI staining. Right: Average number of fluorescent puncta in chloroquine-injected relative to PBS-injected livers. n = 6 (PBS) and 7 (CQ) sections (total 740 cells) coming from three independent experiments. e, f Immunostaining for LAMP1 (e) or for CD68 or α-SMA (f) to label Kupffer and hepatic stellate cells, respectively, in fed or 24 h starved KFERQ-Dendra mice livers. Insets: green channel at higher magnification. g Quantification of Dendra positive puncta in hepatocytes, Kupffer and stellate cells in fed mice livers (left), comparison in fed and starved mice livers (middle) and fold changes in Dendra-positive puncta with starvation (right). n = 13 (fed hepatocytes), 15 (starved hepatocytes), 17 (fed and starved Kupffer cells), 15 (fed stellate cells), and 12 (starved stellate cells) sections coming from three independent experiments. Data are presented as mean values ± s.e.m. Two-tailed unpaired t test (d), One-way ANOVA followed by Tukey’s multiple comparisons tests (g left and right) and Two-way ANOVA test followed by Sidak’s multiple comparisons tests (g middle) were used. Test statistic (F, t, R) and freedom degree for (d) and (g) and source data are provided in the Source Data file.