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. 2019 Jun 11;9:8462. doi: 10.1038/s41598-019-44934-5

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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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Endogenous TARDBP regulates HBV gene expression in a HBV infection model. (a) NTCP-HepG2 C4 cells were transduced with a lentiviral vector allowing the expression of a control shRNA (shControl) or shRNA directed against TARDBP (shTardbp). After selection with puromycin, reduction of TARDBP mRNA and protein level by shRNA was confirmed by qPCR and western blotting, respectively. (b) The cells in (a) were then infected with HBV in duplicate sets for 12 days. Supernatants were collected at the indicated time points for extracellular HBV DNA analysis. (c) A separate portion of supernatants from (b) were also analyzed by CLEIA for measurement of HBs and HBe antigen levels. (d) To detect the level of the HBV core protein, one set of the cells were harvested at the end of the 12-day infection period for protein lysis and subjected to western blotting using the anti-HBc antibody. (e) For detection of the core-associated HBV DNA, the lysates in (d) were subjected to immunoprecipitation by an anti-HBc antibody followed by qPCR (left panel) and Southern blotting (right panel). (f) Total RNA was extracted from the other set of cells from after the 12-day infection period in (b) and subjected to RT qPCR to detect HBV precore, pregenomic (pg) and total mRNAs. Gene expression was normalized to that of GAPDH. The data is shown as the mean ± SD (n = 3 per bar), **P < 0.01, ***P < 0.001 and ns, non-significant.