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. 2023 Jul 13;25(8):1101–1110. doi: 10.1038/s41556-023-01178-w

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© The Author(s) 2023

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 — a, Cells expressing endogenously tagged spartin (with mScarlet-I) and transiently expressing LAMP1–mNG reveal spartin and LAMP1 co-localization after oleate withdrawal. SUM159 cells treated with 0.5 mM OA for 24 h (top) and chased for 3 h after OA withdrawal (bottom). Scale bars: full-size, 20 μm; insets, 2 μm. b, Overlap of spartin and LAMP1 in cell periphery with or without OA withdrawal for 2 h after 18 h OA addition as shown in a (Pearson’s coefficient analysis). Mean ± s.d., n = 6 fields of view, three independent experiments, **P = 0.0091, two-tailed unpaired t-test. c, WT or spartin KO cells transiently co-expressing HaloTag–LiveDrop (pre-labelled with 100 nM JF549) and LAMP1–mNG reveal spartin deficiency impairs LD targeting to lysosomes. Cells treated with 0.5 mM OA (24 h) and chased for 3 h after OA withdrawal. Scale bars as in a. d, Overlap of LiveDrop and LAMP1 in cell periphery shown in c quantified by Pearson’s coefficient analysis. Mean ± s.d., n = 12 cells (WT) and 14 cells (KO) from three independent experiments, ****P < 0.001, two-tailed unpaired t-test. e, Spartin interacts with LC3A and LC3C. HEK293T cells transiently co-expressing HA-spartin together with 3xFLAG–ATG8 (LC3A, LC3B, LC3C, GABARAP, GABARAPL1 or GABARAPL2) were subjected to immunoprecipitation with anti-FLAG antibody and analysed by immunoblot with anti-HA and anti-FLAG antibodies. f, Spartin co-localizes with LC3A. Cells expressing mScarlet-I–spartin full-length and HaloTag–LC3A (pre-labelled with 100 nM JF646) were treated with 0.5 mM OA for 24 h and chased for 3 h after OA withdrawal. Scale bars: full-size, 20 μm; insets, 2 μm. g, Delivery of spartin-coated LDs to lysosomes is autophagy-dependent. Co-localization analyses between transiently overexpressed mScarlet-I–spartin and LAMP1–mNG in SUM159 cells lacking ATG5, ATG7 or FIP200. Scale bars: full-size, 20 μm; insets, 2 μm. h, Overlap of spartin and LAMP1 in cell periphery shown in g (Pearson’s coefficient analysis). Mean ± s.d., n = 6 cells (WT), 11 cells (FIP200 KO), 13 cells (ATG7 KO) and 3 cells (ATG5 KO) cells from three independent experiments, ****P < 0.001, one-way analysis of variance, Dunnett’s multiple comparisons test. Source numerical data and unprocessed blots are available in source data.

Source data