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. 2023 Jul 19;8:275. doi: 10.1038/s41392-023-01487-4

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© The Author(s) 2023

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. 5 — ATF1 depletion impairs mitochondrial rejuvenation. a Representative images of the mitochondria in iDox-shATF1 MDA-MB-231 (Dox −/+) and iDox-shATF1-ATF1 rescued cells under the electron microscope. Bar graphs show the results from the morphometric analysis of cristae number or cristae length/mitochondrial area in cells (n = 20 mitochondria/group; mean ± SD). Scale bar, 0.2 μm. b Mitochondrial ROS as indicated by flow cytometry analysis of MitoSOX Red. Parental and iDox-shATF1#1/#4 MDA-MB-231 cells were treated with Dox for 3 days or 6 days (n = 3; mean ± SD). c Mitochondrial turnover determined by flow cytometry analysis of cells 48 hr after transiently transfected with mitoTimer (left panel. n = 3; mean ± SD). d Mitochondrial turnover as determined by flow cytometry analysis of cells 48 hr after transiently transfected with mitoTimer in iDox-shATF1 H460, A549 and T47D cells (n = 3; mean ± SD). e Percentages of cells with damaged mitochondria as indicated by MitoTracker Deep-Red^low Green^high cells (n = 3; mean ± SD). For a–e, P values, Tukey’s multiple comparisons after 1-way ANOVA. f Representative plots showing mitochondrial damage as determined by flow cytometry analysis of MitoTracker Deep-Red and MitoTracker Green. The iDox-shATF1#1/#4 MDA-MB-231 cells were treated with Dox for 4 days, challenged with CCCP for 6 h before MitoTracker staining. g Percentages of cells with damaged mitochondria as indicated by MitoTracker Deep-Red^low Green^high cells treated with CCCP in combination with dynein inhibitor (DynI). n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA. h Immunofluorescence of Tom20 and alpha tubulin in iDox-shATF1#1 MDA-MB-231 cells treated with Dox for 4 days. Scale bars, 20 μm. i Left panel, mitochondria localization determined by confocal microscopy using MitoTracker Red staining. Right panel, MitoTracker Red intensity (mean ± SD) as a function of distance to nuclei was analyzed (n = 28 for CT group, n = 52 for Dox group and n = 20 for wild-type rescued group, respectively). Scale bar, 20 μm. j Sphere formation of iDox-shATF1 MDA-MB-231 cells with/without Dox treatment, cells were rescued with MitoTempo (MitoT, 10, 20, 30 μM), and collected for sphere forming assays. k Sphere formation of iDox-shATF1 MDA-MB-231 cells with/without Dox treatment, cells were rescued with MitoQ (0.05 μM), and analyzed for sphere forming capacity (for j–k, n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant