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. 2020 Sep 17;10:15234. doi: 10.1038/s41598-020-71970-3

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© The Author(s) 2020

Open AccessThis 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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EV-A71-infected differentiated IMR-32 cells exhibit complete autophagic flux. Differentiated IMR-32 cells were infected with EV-A71 at an MOI of 2. (A) The autophagy tandem sensor RFP-GFP-LC3B was added to EV-A71-infected differentiated IMR-32 cells and observe the expression of GFP-LC3 and RFP-LC3 at 24 and 40 h postinfeciton. The scale bar = 10 μm. (B) The average number of GFP-LC3 puncta and RFP-LC3 puncta was quantified. (C) Western blotting was performed to measure the protein levels of p62 and EV-A71 3D^pol at indicated time points. (D) The band intensities of p62 were measured using ImageJ software and normalized to the β-actin. The time point of mock-infection 16 h was used as a reference. (E) Differentiated IMR-32 cells were treated with 5 or 10 nM bafilomycin A1 or with only DMSO and were infected with EV-A71 (MOI = 2) or underwent mock infection. Western blotting was performed to measure the levels of LC3-I, LC3-II and EV-A71 3D^pol at indicated time points. (F) The band intensities of LC3-II were measured using ImageJ software and normalized to the β-actin. The time point of 16 h was used as a reference. (G) Differentiated IMR-32 cells were cotreated with 10 μM pepstatin A and 10 μM E-64d or only DMSO and infected with EV-A71 (MOI = 2) or mock infection. Western blotting was performed to measure the protein levels of LC3-I, LC3-II and EV-A71 3D^pol at indicated time points. (H) The band intensities of LC3-II were measured using ImageJ software and normalized to the β-actin. β-actin was used as an internal control in all western blots. The error bars represent the SD. Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001.