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. 2020 May 19;10:8284. doi: 10.1038/s41598-020-64924-2

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© The Author(s) 2020

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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DMB induces mitophagy in H9C2 cells. Differentiated H9C2 cells were treated with vehicle (DMSO) or 1 µM DMB overnight with or without autophagy inhibitor bafilomycin A1 (BafA1, 50 nM). (a) Western blot analysis and quantification of autophagy markers in the cell whole lysate (n = 3 plates/group); (b) Western blot analysis and quantification of p62 in the mitochondria-enriched subcellular fraction of DMSO or DMB-treated cells with or without BafA1 (n = 4 plates/group). The samples derive from the same experiment. The blots were processed in parallel and developed using the same exposure time. (c) Immunofluorescence microscopy of the mitophagy adapter p62/SQSTM1 and the mitochondrial protein TOM70 in DMSO and DMB-treated H9C2 cells, scale bars as indicated; (d) Percentage of cells showing colocalization of p62 and TOM70. (n = 4 plates/group with 10 fields of cells captured for each plate. Scoring was performed blinded. All values are presented as mean ± standard deviation. ANOVA with Tukey posthoc test was used to compare the groups in A and B. Standard Student’s t-test was used to compare the groups in D. WB figures were cropped and all densitometry was performed using NIH ImageJ software v 1.51 (https://imagej.nih.gov/ij/download.html). Full-length blots/gels are presented in Supplementary Figure 3.