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. 2017 Oct 24;8:1129. doi: 10.1038/s41467-017-01186-z

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© The Author(s) 2017

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. 3 — Cholesterol crystal production affected by altering FC-CE abundance. a–c HAoEC were treated for 3 days with 250 μg/ml LDL alone, or in parallel with CI-976 to inhibit the cholesterol ester transferase ACAT, or the cholesterol ester hydrolase DEUP to increase free cholesterol (FC) or cholesteryl ester (CE) content respectively. a Staining of neutral lipids with Oil Red O after 3 days of LDL treatment reveals differences in cellular CE content (scale bar = 100 μm). b, c EC were also analyzed by flow cytometry after being stained with Bodipy to detect CE b, or Filipin to detect FC (c) (n = 3 in duplicates, error bar indicates SEM, * indicates significance to LDL treatment at p < 0.05, *** p < 0.001 in unpaired t-test). d Polarized light microscopy analysis shows an increase in large, plate-shaped crystals after CI treatment, while DEUP treatment results in increased abundance of needle-shaped crystals as compared with LDL-treated control HAoEC. (scale bar = 50 μm) e Treatment of HAoEC with DiI-LDL for 1, 3 or 5 days reveals similar uptake of LDL between groups, although more needle-shaped CC (white arrows) are seen in DEUP-treated cells, while large DiI-negative spaces (yellow arrows) that we believe may be crystalline particles were observed in CI-976-treated cells. (scale bar = 50 μm) f SEM images of HAoEC in vitro after 5 days of LDL treatment reveal production of cholesterol crystals, with CI-976 treatment leading to mainly plate-shaped and DEUP treatment leading to mainly needle-shaped CC. (representative images of n = 4)