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. 2019 Feb 18;9:2186. doi: 10.1038/s41598-019-38497-8

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© The Author(s) 2019

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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Rapid accumulation of galectin-3 around lysosomes upon chemically-induced lysosomal damage. (a) JIMT-1 breast cancer cells were treated with 0.3 mM glycyl-l-phenylalanine 2-naphthylamide (GPN) or 1% (v/v) dimethyl sulfoxide (DMSO) (used as control) for 13 minutes. The samples were then fixed, permeabilized, and stained to visualize nuclei (H33342) and galectin-3. Images are shown as maximum intensity projections of z-stacks obtained using confocal microscopy. Treatment with GPN induces distinct galectin-3 puncta in the cytosolic space of the cells. Scale bars are equal to 10 µm. (b) The mean number of galectin-3 puncta/nucleus from 74 or 92 images of cells treated with DMSO or GPN, respectively, from a total of 20 independent cell cultures for each treatment. The analysis was carried out in ImageJ on maximum intensity projections of z-stacks. The result is presented as mean values ± SEM, and the difference was statistically evaluated by an unpaired two-tailed t-test (****P < 0.0001). (c) JIMT-1 breast cancer cell treated with 0.3 mM GPN for 13 minutes and then stained to visualize lysosomal-associated membrane protein (LAMP)-1 and galectin-3. The images (single optical planes) show clear co-localization of galectin-3 accumulations with LAMP-1, seen best by observing the intensity profiles for the vectors highlighted in white, indicating that the galectins act in the vicinity of GPN-damaged lysosomes. In addition, LAMP-1 is present in many undamaged lysosomes, without galectin-3 accumulation. The images shown are representative of a larger number of images showing similar co-localization patterns. Arrowheads indicate areas of co-localization and have the same xy-coordinates in all images. Scale bars are equal to 10 µm.