The human hepatocarcinoma cell lines H7402 and HepG2 and the T-lymphocyte cell line Jurkat were cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% calf serum (Sijiqing Co, Hangzhou, China) at 37°C in 5% CO₂.

Production of the mAbs against HMBOX1 was performed using a hybridoma technique. As previously described (4), the HMBOX1 fusion protein mixed with acid-treated naked Salmonella minnesota R595 bacteria was used to intraperitoneally immunize 8-week-old BALB/c female mice at 2-week intervals. When the titer of the mouse serum was higher than 1:10⁵, cell fusion and cloning programs were performed. Two hybridoma lines, 2A5F4 and 4A4F2, were obtained, and both stably produced anti-HMBOX1 antibodies. Anti-HMBOX1 mAbs were prepared from the ascetic fluid of the BALB/c mice.

The anti-HMBOX1 antibody was purchased from Abcam Inc. (Cambridge, MA, USA; http://www.abcam.com/HMBOX1-antibody-ab50392.html). It is a rabbit anti-mouse polyclonal antibody that is used in Western blot analysis and ELISA assays. It was used as a positive control Ab in this study.

Cell extracts were separated by SDS-PAGE on a 12% polyacrylamide gel and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting. The membranes were blocked with 5% nonfat milk in TBS/0.1% Tween-20 and then incubated with mAb 2A5F4, mAb 4A4F2 or the control Ab against HMBOX1 for 2 h at room temperature (RT), and subsequently washed with TBST 3 times and incubated for 1 h with anti-murine antibody-conjugated horseradish peroxidase (HRP) (Dako, Glostrup, Denmark) or anti-rabbit antibody-conjugated HRP (Cell Signaling Technology, Danvers, MA, USA). After washing 5 times with TBST, the proteins were detected with the enhanced chemiluminescence system (Pierce Biotechnology, Rockford, IL, USA).

Cell extracts were prepared by resuspending 1×10⁷ cells in ice-cold gentle lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM PMSF, 1 mM NaVO₃, 50 mM NaF, 1% NP-40 and protease inhibitors] for 30 min on ice. The extracts were then clarified by centrifugation (16,000 x g for 15 min at 4°C), and the supernatant was collected. After incubation with protein A-Sepharose beads (GenScript, Nanjing, China) for 10 min at 4°C, the supernatant was cleared and concentrated using the Bradford method while BSA (Bio-Rad Laboratories, Hercules, CA, USA) was used as a standard. The cleared extract was incubated at 4°C overnight with mAb 2A5F2 or mAb 4A4F2 at a dose of 5 μg/500 μg total protein in 500 μl and then incubated with 50 μl protein A-Sepharose beads at 4°C for 1 h. The immune complexes were recovered by centrifugation, washed 3 times with PBS buffer, and solubilized in loading buffer by heating at 100°C for 5 min. Immunoprecipitated complexes were electrophoresed on a 12% reduced SDS polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted as previously described (5).

The hepatic cell lines were adjusted to 1×10⁶ cells/ml, fixed in 4% cold paraformaldehyde for 10 min at RT, and permeabilized with vigorous overtaxing in 500 μl ice-cold 0.1% saponin per 10⁶ cells. The primary antibody against HMBOX1 (mAb 2A5F4 or mAb 4A4F2) was then added at a dilution of 1:50, and the cells were incubated at 4°C overnight. After washing three times with PBS, cells were resuspended in 100 μl PBS and incubated for 1 h with FITC-conjugated anti-mouse IgG antibody at a dilution of 1:100 (eBioscience, San Diego, CA, USA). Fluorescence was assayed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The isotype-matched control sample was included as the standard control.

The immunohistochemical SP staining method was used to detect the HMBOX1 protein levels in hepatocytes growing on glass chips or a tissue microarray (OD-CT-Com03-002; Shanghai Biochip, China). The sections were incubated at 4°C overnight with mAb 2A5F4 or mAb 4A4F2 in a humidified chamber. The mouse SP detection and DAB envision systems (both from Zimbio, Beijing, China) were applied.

To verify the specificity and sensitivity of mAb 2A5F4 and mAb 4A4F2 against HMBOX1, the HMBOX1 fusion protein was gradient diluted and detected by Western blot analysis with mAb 2A4F4 and mAb 4A4F2. As shown in Fig. 1, the lowest concentration of the HMBOX1 protein detected was 0.8 μg/ml. Thus, the specificity and sensitivity of mAb 2A5F4 and mAb 4A4F2 were found to be comparable with that of the commercial HMBOX1 antibody from Abcam Inc.

Previous research has confirmed that HMBOX1 is widely expressed in human tissues (4). Here, the expression profile of HMBOX1 was further identified in various human tumors. As shown in Fig. 2, a tissue microarray was used to analyze the expression of HMBOX1. The expression level of HMBOX1 was found to be dramatically decreased in liver cancer compared with that in adjacent normal tissue, whereas HMBOX1 in kidney tissue was mainly expressed in the renal tubule, and the expression level of HMBOX1 was much higher in clear-cell carcinoma of the kidney originating from the renal tubule. Additionally, there was no significant change in HMBOX1 expression in the other cancer tissues tested. Similar to the results reported by Chen et al (2), high levels of HMBOX1 protein were detected not only in pancreatic cancer but also in adjacent normal tissue.

Cell lines are commonly used as in vitro models in scientific research. Thus, the expression of HMBOX1 was further evaluated in the human hepatocellular carcinoma cell lines H7402 and HepG2 by immunoanalytical methods. First, the HMBOX1 protein was found to be localized in the nucleus and cytoplasm as detected by Western blot and immunohistochemical analyses (Fig. 3A and C). Jurkat, a T-lymphocyte cell line, was used as a positive control. Similar to the control Ab, positive immunostaining for HMBOX1 was observed in both the H7402 and HepG2 cells using mAb 4A4F2 (Fig. 3A), which is consistent with previous reports (4). Second, immunoprecipitation was performed with mAb 2A5F4 and mAb 4A4F2. As shown in Fig. 3B, mAb 2A5F4 was found to immunoprecipitate two different variants of HMBOX1, HMBOX1 (410aa, 47 kDa) and HMBOX1b (304aa, 35 kDa), from the H7402 and HepG2 cells, while mAb 4A4F2 did not. One possible explanation was that the type of mAb 4A4F2 used was IgM (4), which has poor affinity to protein A-Sepharose beads (6), and thus the immune complex could not be precipitated. Finally, mAb 2A5F4 and mAb 4A4F2 were also used for immunohisto chemical (Fig. 3C) and flow cytometric (Fig. 3D) analyses, and as expected no significant difference in expression was noted between the cell lines. These findings demonstrated that the HMBOX1 protein was expressed in H7402 and HepG2 cells, and that these cell lines are suitable for investigating the mechanisms of HMBOX1 in HCC development in vitro.