Clinical samples and immunohistochemistry

Formalin-fixed and paraffin-embedded liver cancer tissues were obtained from patients undergoing surgery at Renmin Hospital, Hubei University of Medicine, ShiYan, China. Six HBsAg-positive HCC tissues and six HBsAg-negative HCC tissues were selected for analysis. The selected pathological tissues were negative for hepatitis A, C, D, and E viruses and negative for human immunodeficiency virus (HIV). Immunohistochemical staining and Western blot analysis were performed as previously described [13]. The antibody used for staining was anti-GP73 (#ab109628, Abcam). The study was conducted according to the principles of the Helsinki Declaration and approved by the Institutional Review Committee of Hubei Medical University according to its guidelines for protecting human subjects. Each participant provided written informed consent.

Xenograft nude mice

Male BALB/cA-nu mice (19.3–23.6 g) aged 5 weeks (n = 6 in each group) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). The tumorigenicity assay in nude mice was performed as described previously [13]. Animal conservation and sacrifice were carried out according to the methods approved by the Animal Care and Use Committee of the Animal Experimental Center of Hubei Medical University.

Cell culture and transfection

Primary human hepatocytes (PHHs) were purchased from the Research Institute for Liver Diseases (Shanghai, China) and cultured as described previously [14]. HepG2 cells, Huh-7 cells, HepG2.2.15 cells, and HEK293T cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). L02 cells and HepG2-NTCP cells were kindly provided by Dr. Jianguo Wu of Wuhan University, China. HepG2 cells, HepG2-NTCP cells, HepG2.2.15 cells, Huh-7 cells, L02 cells, and HEK293T cells were cultured as previously reported [15]. For PHHs, the plasmid was transfected into cells with an Amaxa Mouse/Rat Hepatocyte Nucleofector™ Kit (Lonza, Basel, Switzerland) following the optimized protocol from the manufacturer's instructions. For other cells, Lipofectamine® 3000 (Thermo Fisher Scientific, USA) was employed to transfect cells with plasmid or small interfering RNAs according to the manufacturer's instructions.

Plasmid construction

For overexpressing GP73, specific primers were used to amplify the coding region of GP73 by PCR (forward: 5′-GCG GAA TTC ATG ATG GGC TTG GGA AAC-3′; reverse: 5′-CCG CTC GAG TCA GAG TGT ATG ATT CCG-3′). The GP73 PCR product was inserted into the pCMV-Tag2B or pWPXLD vector. For knockdown of GP73, the following sequences were used to target the human GP73 CDS: 5′-GTTGAGAAAGAGGAAACCAAT-3′ [16].

All constructs were confirmed by DNA sequencing.

Virus production and transduction of cell lines

For HBV infection of HepG2-NTCP cells, the culture supernatant of the HepG2.2.15 cell line was concentrated 100-fold by ultracentrifugation. The concentrated HBV stock titer (genome equivalents/ml, GEq/ml) was assessed using qRT-PCR. The multiplicity of infection (MOI) was defined as the genome equivalents per cell (GEq/cell). HepG2-NTCP cells were infected as described previously [15].

Construction of the stable GP73 knockdown cell line was performed as previously described [13]. Briefly, the lentivirus plasmid pWPXLD (Addgene plasmid #12258) was modified by inserting a T2A peptide between the multiple cloning site (MCS) and the GFP gene. Then, the gp73 gene was amplified by PCR and inserted into the MCS of pWPXLD. After that, lentivirus was produced in HEK293T cells and transfected into Huh7 cells or HepG2 cells. The GFP-positive cells were then enriched by flow cytometric sorting on the basis of GFP expression and named Huh7-GP73 or HepG2-GP73.

Construction of the stable GP73 knockdown cell line was performed as previously described [16]. Briefly, short hairpin sequences targeting gp73 were inserted into the pLKO.1-TRC vector (Addgene #10879). After that, lentivirus was produced in HEK293T cells. Concentrated shRNA lentiviruses were used to transfect Huh7-GP73 cells or HepG2-GP73 cells. Transfected cells were cultured in puromycin (2.5 μg/ml) selection medium for at least 7 days to establish stable GP73 knockdown cell lines, which were named Huh7-GP73-RNAi or HepG2-GP73-RNAi.

ELISA, Western blotting and immunofluorescence

For ELISAs, cell culture supernatants were collected to detect the levels of HBeAg and HBsAg with an ELISA kit (Kehua Bioengineering, Shanghai, China).

Western blot analysis was performed as described previously [13]. Antibodies were used to detect HA tags (#SAB2702196, Sigma), FLAG tags (#F7425, Sigma), GP73 (#ab109628, Abcam), NF-κB (p50) (#ab7549, Abcam), IFN-β (#ab180616, Abcam), IFN-λ1 (# MA5-30682, Invitrogen), IL-6 (#ab9324, Abcam), TNF-α (#ab6671, Abcam) and β-actin (#A1978, Sigma). ImageJ (http://rsb.info.nih.gov/ij/) software was employed for band intensity quantification of the Western blot results.

Quantitative RT-PCR analysis

Total RNA was extracted from cells using TRIzol reagent (Invitrogen). RT-PCR primers were shown in Additional file 1: Table S1. Expression level data were normalized to the GAPDH expression level in each sample.

HBV DNA was detected by TaqMan real-time PCR using the following primers: 5′-AGA AAC AAC ACA TAG CGC CTC AT-3′, 5′- TGC CCC ATG CTG TAG ATC TTG-3′ and probe 5′-TGT GGG TCA CCA TAT TCT TGG G-3′.

Flow cytometry

A CytoFlex (Beckman Coulter, USA) or MoFlo flow cytometer (Beckman Coulter, USA) was employed to analyze GFP signals or cell immunophenotyping. Single-cell suspensions were tested by cell immunophenotyping analysis after cells were stained with fluorescently labeled antibodies: CD90-APC (clone 5E10, eBioscience, 1:100), CD133-APC (clone AC133, Miltenyi Biotec, 1:100), and CD117-APC (clone 104D2, eBioscience, 1:100). Flow cytometry data were analyzed using CytExpert software (Beckman Coulter, USA).

Wound healing assay

Cells were plated in 6-well plates. When the cells grew to 80%-90% confluence, the cell monolayers were scraped with a sterile micropipette tip. In the next step, wounded monolayers were gently washed with phosphate buffer solution (PBS) to remove cell debris. At three time points (0, 24 and 48 h), the distance between the two edges of the wound was calculated for three different positions.

Sphere formation assay

Sphere formation assays were performed as previously reported [13]. HepG2-GFP cells and HepG2-GP73 cells were plated in ultralow attachment 6-well plates (Corning, Inc., Corning, NY, USA) at a density of 2000 cells/well. For Huh7-GFP and Huh7-GP73, a single cell was successively sorted into ultralow attachment 96-well plates by a MoFlo flow cytometer (Beckman Coulter, USA).

Statistical analysis

All experiments were performed in triplicate. All data were recorded as the means ± standard deviations (SD) unless otherwise stated. Prism 5 software (GraphPad Software) was used for statistical tests. P < 0.05 was considered statistically significant.