Note that all patients do not have data all the time points

the fold of the open area. Scale bar indicates 400 m. Values are mean SD from three replicates. P<0.01. Trans-well assay to examine cell motility in Hep3B, Huh-7, and SK-hep1 cells pre-treated with 50 g/mL IgG or HS20. Scale bar indicates 50 m. doi:10.1371/journal.pone.0137664.g002 HS20 inhibited HGF-induced cell migration and motility in HCC cells To investigate the underlying mechanism of how HS20 regulates cell migration and motility in HCC cells, we initially detected the effect of canonical and non-canonical Wnt signaling on HCC cell motility. Hep3B cells were treated with Wnt3a- or Wnt5a-conditioned media, but none of them had a significant effect on cell migration. This observation suggests that GPC3 may coordinate with other signaling to regulate cell movement. Several studies report that glypican-1 and glypican-4 are involved in HGF-dependent signaling. Therefore, we detected whether HGF is expressed in HCC cells. As shown in Fig 3A, Hep3B, Huh-7, and SK-hep1 cells all expressed endogenous HGF. We knocked down HGF with siRNA; at least 70% of HGF expression was reduced after transfection. The HGF 6 / 13 Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Fig 3. HGF knockdown reduced the inhibitory effect of HS20 in HCC cells. Western blots to examine the expression of HGF in Hep3B, Huh-7 and SKhep1 cells. Western blots to examine the knockdown efficiency of HGF. Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 g/ mL human IgG or HS20. Cell migration ability was then measured with a wound healing assay. The open wound area at 0 hours was regarded as 100%. Scale bar indicates 400 m. Values represent mean SD from three replicates. P<0.01 compare to IgG group. Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 g/mL human IgG or HS20. A Trans-well assay was performed to examine cell motility. The OD590nm value of control siRNA group with IgG treatment was set up as 100%. Scale bar indicates 50 m. Values are mean SD from three replicates. P<0.1 and P<0.01 compared to the IgG group. doi:10.1371/journal.pone.0137664.g003 knockdown cells had a slower cell migration rate. When treated with HS20, cell migration of HGF knockdown cells was not significantly inhibited. Similarly, HGF knockdown cells exhibited reduced cell motility. HS20 slightly inhibited cell motility in HGF knockdown cells but the inhibition was significantly less than that of control cells. All of these observations indicated that HGF regulated HCC cell migration and motility. Moreover, we treated the cells with purified recombinant HGF and then examined cell migration and motility. We found that HGF stimulation caused dramatically faster cell migration and motility, and when we pre-treated cells with HS20, the increase in cell migration was blocked. MedChemExpress KU55933 pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed/19736794 In a cell motility assay, HS20 abolished HGF-driven cell motility in both Hep3B and Huh-7 cells, whereas cells treated with a control antibody did not exhibit significant changes. These observations indicated that, by neutralizing the function of HS chains on PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19737141 GPC3, HS20 blocked HGF-mediated cell migration and motility of HCC cells. The HS chains of GPC3 were involved in HGF/Met activation To evaluate whether the HS chains of GPC3 play an important role in HGF activation, we examined the interaction of GPC3 and HGF. We incubated purified GPC3 or mutant GPC3 without HS chains with recombinant HGF to perform a pull down assay. As shown in Fig 5A, GPC3 interacted with HGF but not with th