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. 2019 Dec 27;9:19890. doi: 10.1038/s41598-019-56535-3

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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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hBD3 inhibited autophagy in intestinal epithelial cells. The intestinal epithelial cells were respectively incubated with rapamycin (200 nM for IEC-6 or 50 nM for Caco2, 24 h) or hBD3 (5 μg/ml, 12 h) as indicated. The expression of p62, Beclin1 and LC3 in each group was determined by western blotting (A), and p62 levels of IEC-6 were also assessed by immunostaining (B, scale bar, 50 μm). IEC-6 cells were infected with Ad-mRFP-GFP-LC3(MOI = 1000) for 48 h and photographed with the confocal microscopy. Representative mRFP-LC3, GFP-LC3 and merge images were shown (C, scale bar, 20 μm). GFP-LC3 dots and mRFP-LC3 dots per cell were respectively counted (D) the number of yellow (autophagosome) and free red dots (autolysosomes) per cell was quantified. (E) The ultrastructure of IEC-6 and Caco2 cells was presented by TEM (F, arrows indicated degradative autophagic vacuoles (AVd), scale bar, 1 μm). Data was expressed as mean ± SD of three independent experiments. Abbreviations: hBD3, human beta defension-3; MOI, multiplicity of infection; TEM, transmission electron microscopy. **p < 0.01, ***p < 0.001.