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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. 2 — Cystinosin deficiency alters autophagy in proximal tubules of mouse and zebrafish kidneys. a Representative confocal micrographs and quantification of numbers of LC3⁺ (left panel; red) or SQSTM1⁺ structures (right panel; red) in LTL⁺ (green) proximal tubules in Ctns mouse kidneys (n = 30 proximal tubules pooled from three kidneys per group; each point representing the number of LC3⁺ or SQSTM1⁺ vesicles in a proximal tubule; two-tailed unpaired Student’s t-test, ***P < 0.001 relative to Ctns^+/+kidneys). Nuclei counterstained with DAPI (blue). b Western blotting and densitometric analyses of LC3 protein levels in whole kidney lysates from Ctns mice (two-tailed unpaired Student’s t-test, *P = 0.029 relative to Ctns^+/+ kidneys, n = 3 mice per group). c Representative images of ctns^+/+ and ctns^del8/del8 zebrafish embryos at 5 dpf and d quantification of cystine levels by HPLC in ctns^+/+ and ctns ^del8/del8 zebrafish embryos at 5 dpf (two-tailed unpaired Student’s t-test,***P < 0.001 relative to ctns^+/+embryos, n = 8 ctns^+/+ zebrafish and n = 7 ctns ^del8/del8 zebrafish). e Western blotting and densitometric analyses of LC3 protein levels in pronephric tubules from 3-month-old ctns^+/+ and ctns ^del8/del8 zebrafish (n = 4 per group; two-tailed unpaired Student’s t-test, *P = 0.03 relative to ctns^+/+ kidneys). f Representative electron micrographs and quantification of numbers and size of autophagic vacuoles in pronephric tubules of ctns^+/+ and ctns^del8/del8 zebrafish embryos at 5 dpf. White squares contain images at higher magnification. Yellow arrowhead indicates autophagic vacuoles. Number of autophagic vesicles: n = 43 (ctns^+/+) and n = 42 (ctns^del8/del8) randomly selected micrographs were pooled from 11 ctns^+/+ and 12 ctns^del8/del8 zebrafish. Average vesicle size: n = 32 (ctns^+/+) and n = 33 (ctns^del8/del8) randomly selected micrographs were pooled from 11 ctns^+/+ and 12 ctns^del8/del8 zebrafish. Two-tailed unpaired Student’s t-test, ***P < 0.001 and ^#P < 0.0001 relative to ctns^+/+ zebrafish. β-Actin was used as a loading control in b and e. Plotted data represent mean ± SEM. Scale bars are 50 μm in a and 5 μm in f