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. 2018 Jan 11;9:161. doi: 10.1038/s41467-017-02536-7

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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. 1 — Abnormal lysosome dynamics and autophagy in CTNS-deficient PT cells. a Left: confocal microscopy and three-dimensional (3D) reconstruction of Ctns mPTCs labeled with anti-LAMP1 (red) antibody. Right: quantification of changes in vesicle size (top, each point representing the average size of LAMP1⁺ vesicles in a cell) and lysosome positioning (bottom, percent of perinuclear or peripheral distribution) (n = 30 cells pooled from 3 Ctns kidneys per group; two-tailed unpaired t-test, ^#P < 0.0001 relative to Ctns^+/+ mPTCs). b–f Ctns mPTCs were cultured in normal growth (Fed; 8 h) or nutrient-deprived cell medium (Starved; 8 h). b Representative confocal micrographs (left) and quantification (right) of numbers of LC3⁺ structures (green) in Ctns mPTCs (n = 100 cells pooled from three Ctns kidneys per group; each point representing the number of LC3⁺ vesicles in a cell; one-way analysis of variance (ANOVA) followed by Bonferroni’s posthoc test, ***P < 0.001 relative to Ctns^+/+ mPTCs under fed conditions; NS, not significant.). c Western blotting and densitometric analyses of LC3 protein levels. β-Actin was used as a loading control. Two-tailed unpaired Student’s t-test, *P < 0.05 relative to Ctns^+/+ mPTCs under fed conditions, n = 3 independent experiments. d Representative electron micrographs (left) and quantification (right) of numbers of autophagic vacuoles per cell (n = 10 micrographs per each condition; one-way ANOVA followed by Bonferroni’s posthoc test, **P < 0.01 and ^#P < 0.0001 relative to Ctns^+/+ mPTCs under fed conditions; NS, not significant). e Representative confocal micrographs and quantification of SQSTM1⁺ structures (red) in Ctns mPTCs (n = 100 cells pooled from three Ctns kidneys per group; two-tailed unpaired Student’s t-test, ^#P < 0.0001 relative to Ctns^+/+ mPTCs). f Representative western blotting of SQSTM1 in Ctns mPTCs. GAPDH was used as a loading control, n = 3 independent experiments. Plotted data represent mean ± SEM. Nuclei are counterstained with DAPI (blue). Scale bars are 10 μm in a, b, and e, and 2 μm in d. Unprocessed scans of original blots are shown in Supplementary Fig. 13