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. 2017 Jan 19;8(1):e2560. doi: 10.1038/cddis.2016.493

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Copyright © 2017 The Author(s)

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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HBx enhances the stem cell-like properties and tumorigenic potential of OV6⁺ liver CSCs. (a) The percentage of OV6⁺ cells among the SMMC-7721 and Huh7 cells stably expressing either LV-HBx or LV-Con was measured via flow cytometry and presented as a dot plot analysis. Representative images are shown in the left panel. Experiments were performed in triplicate, and all data are shown as the mean±S.D. *P<0.05 and ***P<0.001. (b) The mRNA expression levels of multiple stemness-related genes in magnetically sorted LV-HBx OV6⁺ or LV-Con OV6⁺ cells from SMMC-7721 and Huh7 cultures were evaluated by qRT-PCR. The fold change was determined using the delta-delta Ct method. Quantified mRNA levels were normalized to β-actin and presented relative to the controls. The data are presented as the mean±S.D. **P<0.01 and ***P<0.001. (c) Representative images of primary and secondary passages of HCC spheres derived from magnetically sorted HCC-LV-HBx OV6⁺ and LV-Con OV6⁺ cells are shown (Scale bar=50 μm; upper panel), and the number of tumor spheroids was counted (lower panel). Experiments were performed in triplicate, and results display the mean±S.D. *P<0.05, **P<0.01 and ***P<0.001. (d) HCC cells infected with either LV-HBx or LV-Con were treated with 5 μM pirarubicin for 4 days, and the percentage of OV6⁺ cells were detected via flow cytometry. Representative results from three independent experiments are shown. (e) OV6⁺ HCC cells were treated with 5 μM pirarubicin, and cell viability was measured by using the CCK-8 assay at the indicated time points. **P<0.05 and ***P<0.001. (f and g) Male NOD/SCID mice were subcutaneously injected with the indicated number of OV6⁺ HCC cells infected with either LV-HBx or LV-Con, and the tumor incidence in the mouse xenografts was evaluated. Representative subcutaneous xenograft tumors from mice injected with a gradient series of lentivirus-infected OV6⁺ cells after 6 weeks of transplantation are shown. The percentage of CSCs present was evaluated 6 weeks after injection by a limiting dilution assay