1. Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor 
Runping Gao, David R. Brigstock 
Gastroenterology 
Volume 129, Issue 3, Pages (September 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Key characteristics of primary cultures of rat PSCs. Micrographs of cytoplasmic oil droplets detected by (A and B) oil red O staining or (C) phase-contrast microscopy in PSCs 3 days after seeding. Immunocytochemistry was used to show the presence of (D) vimentin, (E) desmin, (F) α-SMA, or (G) CCN2 in cultures of activated PSCs. Reverse-transcription polymerase chain reaction analysis showed a progressive increase in expression of (H) FN, collagen I, TGF-β1, or CCN2 as a function of time in primary culture, as compared with consistent expression of β-actin. (Original magnification: A and G, 10×; D–F, 20×; B and C, 40×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Regulation of CCN2 production in PSCs. (A) PSCs were metabolically labeled with [35S]cysteine/methionine for 6 hours in the absence or presence of 20 ng/mL TGF-β1. The major 38-kilodalton CCN2-immunoreactive band detected in the medium and cell lysates is indicated. Densitometry of the bands showed that TGF-β1 significantly increased CCN2 levels as compared with controls (★★P < .01). (B) Activated PSCs were transfected with either pTA-Luc (pTA) or pCCN2 promoter-Luc (pCCN2) for 3 hours and cultured under serum-free conditions for another 6 or 24 hours in the absence or presence of 20 ng/mL TGF-β1, 10 ng/mL PDGF, 25 mmol/L ethanol, or 200 μmol/L acetaldehyde. Luciferase enzyme activity in cell lysates was quantified using a luciferase reporter assay kit. **P < .01 vs pCCN2 group at 6 hours; ★★P < .01 vs pCCN2 group at 24 hours; #P < .05 vs other stimulators at 24 hours.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Adhesion rat activated PSCs to CCN2 through cell surface integrin α5β1 and HSPGs. (A) PSCs were preincubated in serum-free medium for 30 minutes in vehicle buffer (No add) or EDTA (5 mmol/L) either alone or in combination with Ca2+ (10 mmol/L) or Mg2+ (10 mmol/L) or Mn2+ (10 mmol/L). The cells were placed into microtiter wells coated with CCN2 and incubated at 37°C for 20 minutes. Wells were then washed and adherent cells were fixed, stained with CyQUANT GR dye, and quantified by measuring the fluorescence intensity at 485 nm excitation/530 nm emission. (B) PSC adhesion assays were performed on microtiter wells that had been precoated with CCN2 following a 30-minute preincubation of the cells with the indicated anti-integrin subunit monoclonal antibody. (C) PSCs were preincubated with anti-integrin α5β1 monoclonal antibody (25 μg/mL) or mouse normal IgG (25 μg/mL) for 30 minutes before adding the cells to the wells that had been precoated with CCN2 or FN. (D) Microtiter wells were coated with CCN2 (2 μg/mL) or FN (2 μg/mL) before the addition of PSCs that had been preincubated at 37°C for 30 minutes with heparin (2 μg/mL) or heparinase I (2 U/mL) or that had been cultured in the presence of 10 mmol/L NaClO3 for the preceding 48 hours, either in the absence or presence of 10 mmol/L Na2SO4. ★★P < .01 vs control; **P < .01 vs “No Add” group; ##P < .01 vs EDTA-treated group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Integrin α5β1 expressed in activated PSCs. Anti-integrin β1 immunoprecipitates (IP) of lysates from activated PSCs were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis under nonreduced condition and Western blot analysis using anti-integrin α5 antibody. The 145-kilodalton immunoreactive protein detected on Western blot corresponds to the predicted size of the integrin α5 subunit.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
CCN2 binds to integrin α5β1 directly. (A) Integrin α5β1 was incubated with CCN2 in Nonidet P40 buffer before treatment with immunoprecipitating polyclonal rabbit anti-CCN2 antibody or normal IgG. Samples were separated on 8% sodium dodecyl sulfate/polyacrylamide gel electrophoresis under nonreduced conditions and transferred onto a nitrocellulose membrane that was immunoblotted with anti-human α5β1 before detection using the enhanced chemiluminescence system. (B) Microtiter wells were precoated with either CCN2 at the indicated concentrations or FN at 2 μg/mL. The wells were blocked and then incubated with 1 μg/mL integrin α5β1 in blocking solution. The plate was developed by addition of anti-human α5β1 monoclonal antibody followed by link antibody and HRP-conjugated streptavidin. The color reaction was developed using HRP substrate measured at A450. ★P < .05 and ★★P < .01 vs control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
CCN2 induces migration, proliferation, and/or DNA synthesis in PSCs. (A) PSC migration assays were performed by placing the cells in culture inserts followed by incubation in a 12-well companion plate for 6 hours in the absence or presence of desired concentrations of CCN2 in the lower chamber. (B) PSCs were preincubated with anti-integrin α5β1 (25 μg/mL) or mouse IgG (25 μg/mL) for 30 minutes. Cell migration assays were then performed in the absence (control) or presence of CCN2 (100 ng/mL) or FN (100 ng/mL) in the lower chamber. (C) Migration assays were performed in the presence of no added factors (control), CCN2 (100 ng/mL), FN (100 ng/mL), PDGF (10 ng/mL), or TGF-β1 (20 ng/mL) in the lower chamber. PSC proliferation was evaluated on cells maintained in 0.1% FBS/DMEM that were stimulated for 36 hours with CCN2 (100 ng/mL), FN (100 ng/mL), PDGF (10 ng/mL), or TGF-β1 (20 ng/mL). DNA synthesis assays were conducted in the same manner except that 0.2 μCi/well [3H]thymidine was added to the media for the final 24 hours of stimulation. ★P < .05 and ★★P < .01 vs control; ★★P < .01 vs “No add” group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
Effect of CCN2 on synthesis of collagen I in PSCs. Activated PSCs were cultured in serum-free DMEM for 24 hours and were then incubated in 1 mL cysteine/methionine-free DMEM containing 1% FCS and 100 μCi/mL [35S]cysteine/methionine for 24 hours in the absence or presence of (A) 50 ng/mL CCN2 or (B) 20 ng/mL TGF-β1. Collagen I in cell lysates or medium was detected by immunoprecipitation. For inhibitory studies, PSCs were preincubated for 30 minutes with 25 μg/mL anti-α5β1, anti-CCN2, or normal IgG before treatment with CCN2 or TGF-β1. ★P < .05 vs control; *P < .05 vs (A) “CCN2” alone group and (B) “TGF-β1” alone group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions