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. 2017 Oct 9;6(10):e387. doi: 10.1038/oncsis.2017.85

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Copyright © 2017 The Author(s)

Oncogenesis is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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Reduction of CMA activity during hypoxia/TGFβ/TNFα induced EMT. (a–c) Induction of EMT in MCF-10A cells by hypoxia (a), TGFβ (b, 10 ng/ml) or TNFα (c, 10 ng/ml) treatment for 10 days. Phase-contrast morphology observation (left) and immunoblots for the indicated proteins (right) are shown. Scale bar represents 20 μm. (d, e) MCF-10A cells were stably infected with KFERQ-PA-mCherry1 and were stimulated with hypoxia/TGFβ/TNFα for EMT induction. Then, cells were photoactivated and maintained under serum starvation for 20 h to fully activate CMA. (d) Representative images of KFERQ- PA-mCherry1 puncta number and distribution in epithelial and mesenchymal MCF-10A cells induced by different treatments. Nuclei are labeled with DAPI. Scale bar represents 20 μm. (e) Quantitative analysis of the number of puncta per cell in >20 cells in at least eight different fields. *P<0.05 and **P<0.01 as compared with the puncta number in epithelial 10A-Con cells. (f) Immunoblots analysis of parental MCF-10A cells and MCF-10A cells induced by hypoxia, TGFβ and TNFα, respectively, for indicated proteins. (g) Left: immunofluorescence staining of LAMP2 together with re-staining of DAPI in parental MCF-10A cells and MCF-10A cells induced by hypoxia, TGFβ and TNFα, respectively. Scale bar represents 20 μm. Right: the distance of LAMP2 from the nucleus were measured using ImageJ. Twenty cells in each group were analyzed. Bar graphs represent the mean±s.d. The P-value was calculated using Student’s t-test. ***P<0.001 as compared with Con group.