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. 2019 Sep 20;10(10):695. doi: 10.1038/s41419-019-1950-1

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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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Fig. 4 — a Tomato-LC3 fusion protein is mostly diffuse throughout the cytoplasm of untreated cells, while punctate staining, representing autophagosomes, are observed when MDA-MB231 cells are treated with CP-31398, or chloroquine (CQ; used as positive control). Scale bars: 20 μm. b CP-31398-induced autophagy was quantified by flow cytometry using CYTO-ID green, which exhibiting bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autophagolysosomes. The upper number indicates the percentage positive cells, and the lower number the mean ± s.d. of three independent experiments. c, d Expression of the phosphatidylethanolamine-conjugated form of LC3 (or LC3-II) (c) and the LC3-II/LC3-I ratio (d) increase following CP-31398 (48 h) treatment of MDA-MB231 cells. e Effect of CP-31398 or chloroquine (CQ) on autophagosomes formation observed by electron microscopy. N nucleus, black arrows autophagosomes. f, g The inhibitor of p53 transcriptional activity PFT-α decreases CP-31398-induced autophagosomes formation, measured by flow cytometry using CYTO-ID green (f) or by counting the number of LC3-Tomato + puncta per cell in fluorescence microscopy pictures (g). *P < 0.05 (unpaired two-tailed Student's t test). h The inhibition of p53 expression using siRNA strongly decrease CP-31398-induced LC3 lipidation (LC3-II) measured by western blot. The normalized LC3-II/Actin ratio was calculated by densitometry and display. Data are representative of three independent experiments (a, c, e, h) or are the mean ± s.d. of three independent experiments (b, d, f, g)