370 Gut and Liver, Vol. 5, No. 3, September 2011 previously.36 Cells (4.5×104) in 6-well culture dishes were inocu- lated on FN-coated or uncoated dishes with 10% FBS supple- mented with Iscove’s MDM for 24 hours, after which a medium containing 100 μM NS398 was used in some cases. After 1 hour of incubation, 100 μM BrdU cell-labeling reagent was added. The cells were fixed after 24 hours, and the percentage of BrdU- labeled nuclei was determined by counting >100 nuclei in three random fields (×400 magnification). 5. Western blot analysis Enhanced chemiluminescence Western blotting detection re- agents were purchased from Amersham Life Science (Arlington Heights, IL, USA), and a monoclonal mouse anti-COX-2 anti- body was purchased from PharMingen (San Diego, CA, USA). Polyclonal rabbit anti-c-Met, -CTGF, -FN, -AKT, -p-AKT, -ERK 1/2, -p-ERK 1/2, and -β-actin antibodies were purchased from Santa Cruz Biotechnology. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). 6. Regulation of COX-2 expression by NS398 and HGF HOCs were selected to evaluate the inhibitory growth effect of the COX-2 inhibitor NS398 (Cayman Chemicals, Ann Arbor, MI, USA). COX-2 expression was inhibited in HOC cultures treated with 100 μM NS398, as described previously.37 Cells (3×105) were grown in Iscove’s MDM supplemented with 10% FBS, 100 μg/mL penicillin, 20 μM HGF, and 0.25 μg/mL streptomycin and then stimulated with 20 μM HGF for various time periods. 7. Statistical analysis All experiments were repeated a minimum of three times. All RT-PCR, BrdU staining, and Western blot data are expressed as the mean±SD. The data in some figures are from a representa- tive experiment, which was qualitatively similar to replicate experiments. Statistical significance was determined by compar- ing the two groups using a two-tailed Student’s t-test. Asterisks in the figures indicate significant differences (p≤0.05) between experimental groups and the corresponding control. RESULTS 1. COX-2 gene and protein expression in activated HOCs HOCs appeared as dark blue areas in hematoxylin and eosin-stained liver sections, due to their large nuclei and small amount of cytoplasm, with numerous HOCs in the periportal region on day 9 after 2-AAF/PHx treatment (Fig. 1A). HOCs were confirmed by labeling with OV-6 (Fig. 1B). An additional immunomarker widely utilized in HOC labeling, Thy1.1, was also used (Fig. 1C and E). These results confirm that the 2-AAF/ PHx model can be used effectively to activate HOCs in peri- portal regions during liver regeneration, consistent with previ- ous observations.38 Additionally, COX-2-expressing cells were evident around the periportal region (Fig. 1D and F), and COX- 2 staining in the HOCs was detected in the cytoplasm (Fig. 1F, 2D, 2G, 2J, and 2M). Double-immunofluorescence staining was performed on serial liver sections to verify COX-2 expression in the HOCs and to assess whether the HOCs co-expressed Thy1, c-Met, EpCAM, and α-SMA with COX-2 (Fig. 2). Fig. 2F and I show that in some HOCs COX-2 (Fig. 2D and G) was colocal- ized with the HOC markers Thy1 (Fig. 2E) and c-Met (Fig. 2H), whereas the co-localization of COX-2 (Fig. 2J) and EpCAM (Fig. 2K) did not occur in the peri-bile ductile or hepatic lobule area (Fig. 2L). Moreover, the co-localization of COX-2 (Fig. 2M) with the myofibroblast marker α-SMA (Fig. 2N) was not observed (Fig. 2O). These results indicate that Thy1+ HOCs expressed COX-2 and c-Met but not EpCAM or α-SMA. The next experiment assessed COX-2 expression in total RNA extracted from normal liver, 2AAF/PHx-treated liver (on day 9), and PHx-treated liver (after 12 hours), and sorted Thy1+ HOCs by RT-PCR (Fig. 3A). COX-2 expression was not detected in normal liver, but was detected in the activated HOC model (2AAF/PHx), the liver regeneration model (PHx), and the sorted Thy1+ HOCs, suggesting that COX-2 participates in HOC activa- tion during liver regeneration. Furthermore, COX-2 and c-Met (HGF receptor) expression was examined in the activated HOC model by Western blotting. Normal liver did not induce COX-2 expression, but HOC induction resulted in a progressive increase in COX-2 expression on days 3-13 after 2-AAF/PHx (Fig. 3B). The detection of COX-2 on day 3 after 2-AAF/PHx suggests that the COX-2-induced PG pathway plays a role in HOC acti- vation. Our immunofluorescence staining results revealed that the HOCs expressed COX-2 and the HOC markers (albumin and AFP), but that they did not express CD45. Interestingly, FN was also evident in the HOCs, indicating that it might play a role in HOC activation or proliferation in the 2AAF/PHx liver model (Fig. 3C). 2. Detection of PG receptors in HOCs The finding that COX-2 was expressed in HOCs indicated that a COX-2-mediated PG pathway might be involved in the HOC proliferation mediated by PG receptors. Indeed, PGE2 binds its receptors, designated EP1, EP2, EP3, and EP4, which have been implicated in carcinoma cell proliferation.39,40 RT-PCR was performed for each EP receptor subtype to determine their ex- pression profiles in HOCs (Fig. 4). Tissues from normal rat liver and rat liver after PHx, as well as sorted Thy1+ HOCs expressed all four PG receptors (though faint EP3 expression was noted in the Thy1+ HOCs), indicating the existence of these receptors in Thy1+ HOCs and their possible role in proliferation. 3. Role of COX-2 and ECM in activated HOCs To explore the downstream events in the PG pathway, HOCs were cultured with 100 μM NS398, which suppressed HOC proliferation. CTGF is a downstream mediator of the fibrogenic properties of transforming growth factor (TGF)-β1, which