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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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Establishment of TARDBP-knockout (KO) cells by a CRISPR/Cas9 system. (a) A TARDBP-deficient NTCP-HepG2 cell line (TARDBP KO) was established by the CRISPR/Cas9 system using the two RNA guide sequences shown. (b) Western blot analysis of the NTCP-HepG2 parent cell and the TARDBP knockout NTCP-HepG2 cell line using the antibodies against NTCP, TARDBP, SIRT1 and the control GAPDH is shown. (c) A TARDBP plasmid was transfected into the KO cells for exogenous protein expression. The expression was confirmed by western blotting using the anti-TARDBP and anti-FLAG antibodies. (d) NTCP-HepG2 parent cell, TARDBP KO and TARDBP KO cells complemented with exogenous TARDBP were transfected with the pGL3-CP plasmid for comparison of the core promoter activities. Cell lysates were harvested after 48 hours for the dual luciferase assay as in Fig. 3e. (e,f,g) HBV infection was carried out in the three cells treated as in (c) for 12 days, similar to the experiment in Fig. 1. The supernatants were harvested at the end of the infection period for measurement of extracellular HBV DNA, HBe antigen and HBs antigen levels, respectively. The data is shown as the mean ± SD, *P < 0.05 **P < 0.01, ***P < 0.001.