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. 2017 Jun 7;8:15750. doi: 10.1038/ncomms15750

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Copyright © 2017, The Author(s)

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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(a) Western blot analysis of TFEB in macrophages after trehalose treatment for indicated times. Ponceau S staining is shown as loading control and densitometric quantification from three separate experiments is shown below. (b) TFEB nuclear localization is analysed by immunofluorescence staining after trehalose treatment and graphed as nuclear TFEB intensity (n≥40 cells per group, scale bar, 10 μm). (c) Western blot analysis of other MiTF transcriptional family members (TFE3 and MiTF) after trehalose or chloroquine (CHQ, 10 μM) treatments for indicated doses and times. Ponceau S staining is shown as loading control. (d,e) TFE3 and MiTF nuclear localization was analysed by immunofluorescence staining after trehalose treatment for indicated times and quantified by the intensity of nuclear staining (n≥500 cells per group). (f) Western blot analysis of polyubiquitinated proteins (FK-1 antibody) in detergent-soluble and detergent-insoluble lysate fractions of vehicle (V) or trehalose (T) treated wild-type macrophages. Lanes 3 and 4 were either vehicle or trehalose pretreated for 3 h, and then co-treatment with cholesterol crystals is performed for 12 h. Lanes 5 and 6 were cholesterol crystal-treated for 6 h and then either treated with vehicle or trehalose alone for another 6 h. (g) Densitometric quantification of f from three similar separate experiments. (h) Immunofluorescence images of wild-type macrophages after indicated treatments using DAPI and antibodies against polyubiquitinated proteins (FK-1) and p62 (scale bar, 5 μm). (i) Graphs represent average p62/ubiquitin+ dot numbers and average p62 intensity per cell for immunofluorescence images in h (numbers of quantified cells are shown under each bar). (j) Wild-type macrophages were co-incubated with cholesterol crystals and trehalose (or vehicle); the per cent of caspase 3/7-positive cells was quantified in three independent experiments (numbers of quantified cells are shown under each bar). (k) Wild-type macrophages were treated as indicated and cell culture media were assayed for IL-1β by ELISA (n=3 independent wells for each treatment). For all graphs data are presented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, two-tailed unpaired t-test compared to zero time point or vehicle treatment group.