**2. Results**

#### *2.1. Relationship between Increased Expression of BAFF and BCL10 and Survival during Liver Regeneration*

BAFF levels in liver tissue were significantly increased at 6 h after partial hepatectomy and peaked at 16 h. However, these changes in BAFF levels were not detected in the serum (Figure 1A). As BCL10 is involved in BAFF-mediated signaling, we then evaluated the status of BCL10 in liver tissues. Western blotting (Figure 1B) and corresponding quantitative results revealed that BCL10 expression was significantly enhanced 24 h after partial hepatectomy in regenerative remnant liver tissues. Moreover, IHC confirmed these findings and showed that BCL10 expression was elevated in regenerative remnant liver tissues (Figure 1C).

**Figure 1.** B-cell activating factor (BAFF) and B-cell CCL/lymphoma 10 (BCL10) expression in 70% partial hepatectomy-induced liver regeneration in mice. (**A**) Serum and tissue levels of BAFF were estimated by EIA at the indicated times after 70% partial hepatectomy. Data are presented as means ± SDs, and comparisons were made between each time and time zero. *n* = 6. \* *p* < 0.05, by two-way ANOVA with Tukey's post hoc test. (**B**) Left panel, expression levels of BCL10 at different times in liver tissues from control or 70% partial hepatectomy (PH) groups were determined by western blotting; Acin was used as loading control. Right panel, the quantitative results of BCL10 western blotting. Data are presented as the relative intensity (BCL10/Actin) ± SD. Comparisons were made between the control and PH groups. *n* = 6. \* *p* < 0.05, by Student's *t*-test. (**C**) Tissue BCL10 staining (brown color) of remnant liver tissues. Magnification, 400×. (**D**) Mice were intraperitoneally injected with 100 μg control IgG (Control Ab) or anti-mouse BAFF-neutralizing antibodies (BAFF Ab) at the time of 70% PH. The number of surviving mice was calculated at different times after 70% PH. *n* = 10 per group.

Mice were intraperitoneally injected with 100 μg anti-mouse BAFF-neutralizing antibodies after 70% partial hepatectomy to clarify the role of BAFF expression in liver regeneration. We found that treatment with anti-BAFF-neutralizing antibodies, but not control IgG, caused death in mice that were subjected to 70% partial hepatectomy within 72 h (Figure 1D). These results demonstrated that BAFF was essential for survival during liver regeneration.

#### *2.2. BAFF*/*BCL10 Signaling Plays an Important Role in Hepatocyte Proliferation*

The role of BAFF/BCL10 signaling in hepatocytes is not well defined. Therefore, we used the normal human embryonic liver cell line CL-48 cells [15] to evaluate the BAFF/BCL10 signaling pathway. We first determined the BAFF receptor expression in the CL-48 cells (Figure 2A) via comparing with PBMC, which was used as BAFF receptor positive expression control. The results demonstrated that the BAFF receptor is expressed in CL-48 hepatocytes. CL-48 cells were treated with recombinant BAFF, and BCL10 expression was determined by immunofluorescence staining. BCL10 was visibly upregulated and localized to the hepatocyte nuclei (Figure 2B). BCL10 siRNA was used to knockdown BCL10 to further clarify the role of BAFF/BCL10 signaling (Figure 2C). First, we determined the effects

of BAFF and BCL10 on hepatocyte growth. The results demonstrated that BAFF did not enhance the growth of hepatocytes. However, transfection with BCL10 siRNA significantly inhibited the growth of hepatocytes (Figure 2D). Moreover, flow cytometric analysis showed that transfection with BCL10 siRNA caused a significant arrest of cells in the G2/M phase of the cell cycles (Figure 2E).

**Figure 2.** BAFF/BCL10 signaling in hepatocye cell proliferation. (**A**) The expression of BAFFR mRNA in human CL-48 hepatocytes was determined by q-Reverse Transcription Polymerase Chain Reaction (q-RT-PCR); commercialized human peripheral blood mononuclear cells (PBMC) cDNA was used as the positive control. (**B**) Left panel, human CL-48 hepatocytes were treated without (control) or with BAFF (1 ng/mL) for 1 h, and the expression of BCL10 was determined by immunofluorescence staining; BCL10 was identified as a green signal, and the nucleus was stained with DAPI (blue). Magnification, 400×. Right panel, the number of BCL10 positive cells was counted under high power field (HPF). *n* = 6. \* *p* < 0.05, by Student's *t*-test. (**C**) CL-48 cells were treated with control or with BCL10 siRNA for 24 h; the expression of BCL10 was determined by western blotting. Actin was used as the loading control. (**D**) CL-48 cells were treated with control or BCL10 siRNA for 24 h prior treatment with BAFF (1 ng/mL). At different time points, relative cell proliferation was determined by the Trypan blue exclusion assay. The data are shown as mean ± SD of three independent experiments. Comparisons were made between the BAFF and bcl10siRNA + BAFF groups. \* *p* < 0.05, by Student's *t*-test. (**E**) CL-48 cells were treated as indicated with BCL10 siRNA or BAFF, as described in (**D**). On day 3, the cell cycle phases were determined by propidium iodide staining and FACScan analysis. Populations of cells in the sub-G1, G1, S, and G2/M phases were analyzed and quantified while using the Cell Quest software, and the data represent the means ±SDs of three independent experiments. Between-group comparisons were performed as indicated. \* *p* < 0.05, by Student's *t*-test.

## *2.3. BAFF Promoted Hepatocyte-Mediated Angiogenesis*

The liver is a vessel-rich organ; the capability of hepatocytes to undergo angiogenesis is critical for the maintenance of liver function. TNF-<sup>α</sup>, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Therefore, we next evaluated the role of BAFF in angiogenesis in hepatocytes while using in vitro angiogenesis assays with HUVECs. The results demonstrated that, when compared with the control group, CM from BAFF-stimulated hepatocytes induced gap formation and permeability changes (Figure 3A), migration (Figure 3B), tube formation on matrix gel (Figure 3C), and proliferation (Figure 3D) of endothelial cells. However, none of these effects were observed when CM from BCL10 siRNA-transfected hepatocytes (with or without BAFF treatment) was applied. These results demonstrated that BAFF stimulation might promote hepatocyte-driven angiogenesis.

**Figure 3.** Effects of conditioned medium derived from BAFF-stimulated hepatocytes on angiogenesis. Conditioned medium from BAFF-stimulated CL-48 cells was used for angiogenic function assays. Four experimental conditions were used: control, the conditioned medium was collected from hepatocytes without any treatment; BAFF group, conditioned medium was from BAFF (1 ng/mL)-stimulated hepatocytes; BCL10siRNA+BAFF group, conditioned medium was from BCL10 siRNA-transfected hepatocytes following BAFF stimulation; and BCL10siRNA group, conditioned medium was from BCL10 siRNA-transfected hepatocytes. All conditioned medium was collected after 24 h culture. (**A**) **Left panel**, confluent and Human Umbilical Vein Endothelial Cell (HUVEC) monolayers were treated with conditioned medium for 1 h, and gap formation was determined by phalloidin staining. **Right panel**, conditioned medium from the indicated conditions was tested with HUVEC monolayers for1husing permeability assays. Data are shown as the relative permeability percentages, with the control conditioned medium in lane 1 defined as 100%. Comparisons are shown between the indicated groups. \* *p* < 0.05. *n* = 5, by one-way ANOVA with Tukey's post hoc test. (**B**) Left panel, HUVECs were treated with conditioned medium for 6 h for migration assays, and representative images of migrated HUVECs in each group are shown. Right panel, quantitative results of migrated HUVECs was used to calculate the cell migration area. Comparisons are shown between the indicated groups. \* *p* < 0.05. *n* = 5, by one-way ANOVA with Tukey's post hoc test. (**C**) **Left panel**, HUVECs were treated with conditioned medium for 6 h for capillary tube formation assays, and representative images of tube formation in each group are shown. **Right panel**, quantitative results of tube formation was calculated under high-power fields. Comparisons are shown between the indicated groups. \* *p* < 0.05. *n* = 5, by one-way ANOVA with Tukey's post hoc test. (**D**) HUVECs were treated with conditioned medium for 1–5 days for cell growth determination by Trypan blue exclusion assays. Data are the relative cell growth percentages for the indicated conditions, with the control group in lane 1 defined as 100%. *n* = 5. Comparisons were made between the BAFF and bcl10siRNA+BAFF groups. \* *p* < 0.05, by Student's *t*-test.

#### *2.4. BAFF*/*BCL10*/*NF-*κ*B Signaling in Hepatocytes Enhanced the Expression of Angiogenesis-Related Factors*

Various factors promote angiogenesis. In this study, we used a commercial protein array to identify BAFF/BCL10-activated angiogenesis-related factors in CM from hepatocytes. The results of the array (Figure 4A,B) revealed that MMP-9, FGF4, and IL-8 were upregulated in CM from BAFF-stimulated hepatocytes when compared with those in the control cells and cells transfected with bcl10 siRNA following BAFF stimulation. Thus, BAFF/BCL10 activated the angiogenesis-related factors MMP-9, FGF4, and IL-8 in hepatocytes, resulting in increased levels in CM.

**Figure 4.** BAFF stimulated Matrix metalloproteinase-9 (MMP-9), Fibroblast growth factor 4 (FGF4), and Interleukin-8 (IL-8) expression in hepatocytes. (**A**) Conditioned medium from the three experimental conditions was applied to protein array analysis for identification of angiogenesis-related factors. Four experimental conditions were used: control, the conditioned medium was collected from hepatocytes without any treatment; BAFF group, conditioned medium was from BAFF (1 ng/mL)-stimulated hepatocytes; bcl10siRNA+BAFF group, conditioned medium was from bcl10 siRNA-transfected hepatocytes following BAFF stimulation; and bcl10siRNA group, conditioned medium was from bcl10 siRNA-transfected hepatocytes. All conditioned medium was collected after 24 h of culture. Data shown are representative images of three independent experiments. Significantly altered protein spots are indicated. (**B**) Quantitative results for MMP-9, FGF4, and IL-8. Results are the relative intensity of spots from the angiogenesis protein arrays. *n* = 3. \* *P* < 0.05, by Student's *t*-test. (**C**) CL-48 cells were transfected with an NF-κB binding site-driven luciferase plasmid and then transfected with bcl10 siRNA for 24 h or treated with BAY117082 (100 nM, for 1 h and then depleted the BAY117082 contained medium by twice wish with culture medium), following treatment with recombinant BAFF (1 ng/mL). After 4 h, NF-κB promoter activities were determined. Data are compared with that from lane 1. *n* = 5. \* *P* < 0.05, by one-way ANOVA with Tukey's post hoc test. (**D**) CL-48 cells were transfected with BCL10 siRNA for 24 h or treated with BAY117082 (100 nM, for 1 h and then depleted the BAY117082 contained medium by

twice wish with culture medium), following treatment with recombinant BAFF (1 ng/mL). After 24 h, the protein levels of total MMP-9, FGF4 and IL-8 in the cell culture supernatants were determined by EIAs. Data are compared with that from lane 1. *n* = 5. \* *P* < 0.05, by one-way ANOVA with Tukey's post hoc test. (**E**) CL-48 cells were transfected with BCL10 siRNA for 24 h or treated with BAY117082 (100 nM, for 1 h and then depleted the BAY117082 contained medium by twice wish with culture medium), following treatment with recombinant BAFF (1 ng/mL). After 8 h, mmp-9, fgf4 and il-8 mRNA levels were determined by qRT-PCR. Data are compared with that from lane 1. *n* = 5. \* *P* < 0.05, by one-way ANOVA with Tukey's post hoc test.

BAFF/BCL10 signaling has been found to be involved in NF-κB activation, and MMP-9, FGF4, and IL-8 are regulated by NF-κB. Therefore, we further investigated the roles of BAFF/BCL10/NF-κB signaling in hepatocytes while using NF-κB binding site-driven luciferase assays; the results revealed that BAFF significantly increased NF-κB activity and that the induction of NF-κB was significantly reduced by transfection with bcl10 siRNA or the NF-κB chemical inhibitor BAY117082 (Figure 4C). The results demonstrated the importance of BAFF/BCL10/NF-κB signaling in hepatocytes.

Next, we determined the e ffects of BAFF on MMP-9, FGF4, and IL-8 induction and the role of BAFF/BCL10/NF-κB signaling in determining MMP-9, FGF4, and IL-8 protein (Figure 4D) and mRNA expression (Figure 4E) in hepatocytes. The results demonstrated that BAFF significantly enhanced MMP-9, FGF4, and IL-8 protein and mRNA expression, whereas transfection with bcl10 siRNA and treatment with BAY117082 significantly blocked these changes in MMP-9, FGF4, and IL-8 expression. Accordingly, we concluded that BAFF enhanced angiogenesis in hepatocytes by promoting the expression of angiogenesis-related factors through a pathway involving BCL10 and NF-κB signaling.

#### *2.5. Downregulation of BAFF Reduced Angiogenesis and Hepatocyte Proliferation in a Liver Regeneration Model*

Based on our in vitro study, we clarified the role of BAFF during liver regeneration by the administration of anti-BAFF-neutralizing antibodies to liver regeneration model mice that were subjected to 70% partial hepatectomy. After 48 h, the mice were sacrificed and the remaining liver tissues were dissected to identify the vessels positive for CD31 (Figure 5A; upper panel), an immunohistochemical marker of endothelial cells, and for Ki67 (Figure 5A; lower panel), a marker of cell proliferation. The quantitative result revealed that, when compared to the control group, mice that were treated with anti-BAFF-neutralizing antibodies showed reduced microvessel density (MVD), which corroborates the CD31-positive staining profiles (Figure 5B). Moreover, when compared to the control group, mice that were administered with anti-BAFF-neutralizing antibodies showed reduced Ki67 staining and liver regeneration (Figure 5C). Furthermore, as mentioned above, the level of MMP-9, an important angiogenic factor regulated by BAFF in the in vitro hepatocyte model, was determined in the remaining liver tissues. The results revealed that the administration of anti-BAFF-neutralizing antibodies significantly reduced the MMP-9 levels in liver tissue (Figure 5D). The results demonstrated that BAFF expression was involved in angiogenesis and hepatocyte proliferation in vivo. Please confirm that this is correct.

**Figure 5.** Microvessel density, cell proliferation, andMMP-9 expression in liver tissue of BAFF-neutralizing antibodies treated mice. (**A**) Mice were injected intraperitoneally with 100 μg control IgG or anti-mouse BAFF-neutralizing antibodies after 70% partial hepatectomy. After 48 h, mice were sacrificed, and the remaining liver tissues were dissected and stained for CD31 and Ki67. Magnification, 400×. (**B**) Quantitative results of microvessel density in remaining liver tissue, *n* = 10 per group. \* *p* < 0.05, by Student's *t*-test. (**C**) Quantitative results of Ki67 positive staining cells in remaining liver tissue, *n* = 10 per group. \* *p* < 0.05, by Student's *t*-test. (**D**) MMP-9 protein level in remaining liver tissue was determined by EIA, *n* = 10 per group. \* *p* < 0.05, by Student's *t*-test.

In summary, our findings suggested that BAFF expression was involved in hepatocyte-driven angiogenesis in liver regeneration, as illustrated in Figure 6.

**Figure 6.** Summary of BAFF in liver regeneration. In this study, we found that BAFF was induced in the remaining liver tissue after 70% partial hepatectomy. Since BAFFR was found to be expressed in hepatocytes, we assumed the BAFF activated BCL10 nuclear translocation through BAFFR. BCL10 nuclear translocation subsequently activated NF-κB-dependent gene expression, including cell proliferation, as consistent with a previous study.<sup>11</sup> Interestingly, we found that BAFF may enhance the expression of factors that promote gap formation, permeability changes, migration, tube formation on matrix gel, and endothelial cell proliferation. Moreover, we found the BAFF/BCL10/NF-κB cascade activated the angiogenesis-related factors MMP-9, FGF4, and IL-8. The results demonstrated that BAFF expression was critically involved in hepatocyte-driven angiogenesis during liver regeneration.
