Journal: International Journal of Molecular Medicine

Article Title: CCN1/Cyr61 enhances the function of hepatic stellate cells in promoting the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2017.3356

Figure Lengend Snippet: CCN1 activates HSCs and affects cell function. LX-2 cells were infected with adenovirus AdCCN1 or AdRFP, respectively. (A) Following infection for 24 h, western blot analysis was performed to detect the expression levels of CCN1 in LX-2 cells, LX-2-RFP cells and LX-2-CCN1 cells. (B) Following infection for 72 h, α-SMA and collagen I, which are markers of HSC activation and fibrosis, were detected in the LX-2-CCN1 cells. (C) Expression levels of p-β-catenin, β-catenin, cyclin D1, VEGF, CD31 and CD34 were analyzed. CCN1 antibody (0.5 μ g/ml) was used to neutralize CCN1 in control groups. (D) Viability of LX-2-CCN1 cells was analyzed using MTT assays. (E) Following treatment with RCCN1 (0.6 μ g/ml), LX-2 cells were analyzed using MTT assays. CCN1 antibody (0.5 mg/ml) was used in assays. The final concentrations were 0.5 and 2.5 μ g/ml. (F) Monolayer scratch assay was used to analyze the migration of LX-2-CCN1 cells. * P<0.05 and ** P<0.01. HSCs, hepatic stellate cells; CCN1, cysteine-rich 61; VEGF, vascular endothelial growth factor; SMA, smooth muscle actin; p-, phosphorylated; RCCN1, recombined CCN1; OD, optical density.

Article Snippet: The membrane was then incubated with antibodies against phosphorylated-β-catenin (cat. no 9566, 1:1,000), total-β-catenin (cat. no. 8480, 1:1,000) (both from Cell Signaling Technology, Inc., Danvers, MA, USA), cyclin D1 (cat. no. 60186-1-Ig, 1:1,000), VEGF (cat. no. 19003-1-AP, 1:1,000), CD34 (cat. no. 14486-1-AP, 1:1,000), CD31 (cat. no. 66065-1-Ig; 1:1,000) (all from ProteinTech Group, Inc.), survivin (cat. no. ab76424; 1:1,000 dilution; Abcam, Cambridge, UK), and c-myc (cat. no. MA1-980, 1:1,000; Thermo Fisher Scientific, Inc.), respectively at 4°C overnight.

Techniques: Cell Function Assay, Infection, Western Blot, Expressing, Activation Assay, Control, Wound Healing Assay, Migration

Journal: International Journal of Molecular Medicine

Article Title: CCN1/Cyr61 enhances the function of hepatic stellate cells in promoting the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2017.3356

Figure Lengend Snippet: CCN1 enhances the function of hepatic stellate cells in promoting the viability of HCCs. (A) Western blot analysis was used to detect expression levels of CCN1 in the original CM and 10X concentration CM collected from LX-2 cells infected with AdCCN1 or AdRFP. Expression levels were also detected following treatment with 2.5 and 5 μ g. HepG2 cells were cultured with or without CM. (B) Viability of HepG2 cells, analyzed using MTT assays. * P<0.05 and ** P<0.01 vs. the CM-LX-2-RFP group. (C) HepG2 cells were cultured alone or were co-cultured with different LX-2 cells in a 6-well plate with Transwell inserts. After 5 days, colony formation of HepG2 cells was examined using crystal violet staining (magnification, ×100). (D) Expression of p-β-catenin, β-catenin, survivin, cyclin D1 and c-myc in HepG2 cells were analyzed using western blot analysis. CCN1, cysteine-rich 61; CM, conditioned medium; HCCs, hepatocellular carcinoma cells; p-, phosphorylated; RCCN1, recombined CCN1.

Article Snippet: The membrane was then incubated with antibodies against phosphorylated-β-catenin (cat. no 9566, 1:1,000), total-β-catenin (cat. no. 8480, 1:1,000) (both from Cell Signaling Technology, Inc., Danvers, MA, USA), cyclin D1 (cat. no. 60186-1-Ig, 1:1,000), VEGF (cat. no. 19003-1-AP, 1:1,000), CD34 (cat. no. 14486-1-AP, 1:1,000), CD31 (cat. no. 66065-1-Ig; 1:1,000) (all from ProteinTech Group, Inc.), survivin (cat. no. ab76424; 1:1,000 dilution; Abcam, Cambridge, UK), and c-myc (cat. no. MA1-980, 1:1,000; Thermo Fisher Scientific, Inc.), respectively at 4°C overnight.

Techniques: Western Blot, Expressing, Concentration Assay, Infection, Cell Culture, Staining