*4.4. Immunohistochemical Staining and Quantification*

Slides were rehydrated in phosphate-bu ffered saline (PBS) for 15 min., and endogenous peroxidases were inhibited by treatment with 3% H2O2/methanol for 10 min. at room temperature (25 ◦C). For blocking, 5% nonfat milk/PBS was used for 30 min. at room temperature. Slides were incubated

with anti-BCL10(sc-5273, dilution used 1:50; Santa Cruz Biotechnology, Dallas, TX, USA), Ki67(sc-7846, dilution used 1:200; Santa Cruz Biotechnology), and CD31 (sc-376764, dilution used 1:200; Santa Cruz Biotechnology) antibodies for 16 h at 4 ◦C and then with peroxidase-conjugated secondary antibodies for 1 h at room temperature. The slides were then developed by immersion in 0.06% 3,3-diaminobenzidine tetrahydrochloride (DAB; DAKO, Glostrup, Denmark), followed by counterstaining with Gill's hematoxylin V.

IHC reactions for CD31 and Ki67 were imaged at low magnification (×40) and CD31 or Ki67-positive cells were counted in 10 representative high power fields (×400). Single immunoreactive endothelial cells, or endothelial cell clusters that were separate from other microvessels, were counted as individual microvessels. The mean visual microvessel density for CD31 was calculated. Ki67-positive cells were only counted in hepatocytes, which are with large cells with cuboidal morphology.

#### *4.5. BAFF, Matrix Metalloproteinase (MMP)-9, Fibroblast Growth Factor 4 (FGF4), and Interleukin-8 (IL-8) Determination*

Mouse BAFF levels in serum or tissue lysates, mouse matrix metalloproteinase-9 (MMP-9) levels in tissue lysates, and human MMP-9, FGF4, and IL-8 levels in conditioned medium were determined while using enzyme immunoassay (EIA) kits (R&D Systems, Minneapolis, MN, USA).

#### *4.6. Culture of CL-48 Hepatocytes and Human Umbilical Vein Endothelial Cells (HUVECs)*

Human normal CL-48 hepatocytes were obtained from (Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with nonessential amino acids, l-glutamine, a 2× vitamin solution (Life Technologies Inc., Grand Island, NY, USA), sodium pyruvate, 10% fetal bovine serum, penicillin, and streptomycin (Flow Labs, Rockville, MD, USA). HUVECs were obtained from Cell Applications (San Diego, CA, USA) and then maintained in endothelial cell growth medium (Cell Applications). HUVECs were used at no more than passage 5. All of the cells were cultured at 37 ◦C in a humidified atmosphere of 5% CO2 and 95% air.

#### *4.7. BAFF Protein and Chemical Inhibitors*

Recombinant human BAFF protein and anti-mouse BAFF neutralizing antibody were obtained from R&D Systems. BAY117082 was purchased from Sigma (St. Louis, MO, USA).

#### *4.8. Preparation of Conditioned Medium (CM)*

The CL-48 cells were washed with PBS twice and cultured in 5 mL serum-free DMEM for 24 h before stimulated with recombinant human BAFF protein for 1 h. The CL-48 cells were then washed with PBS twice and cultured in 5 mL serum-free M199 medium for 24 h. CM was then collected and clarified by centrifugation (4 ◦C, 1000× *g*, 5 min.) to remove cell debris. A solution of 25 mM HEPES bu ffer (pH 7.4), 1 mg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.02% NaN3, and 0.1% bovine serum albumin (Sigma) was added to the CM. The CM was finally frozen and stored at −70 ◦C until use.
