1. Volume 59, Issue 6, Pages 2084-2094 (June 2001)
Cell surface heparan sulfate proteoglycans control the response of renal interstitial fibroblasts to fibroblast growth factor-2 
Aled Clayton, Janet Thomas, Gareth J. Thomas, Malcolm Davies, Robert Steadman 
Kidney International 
Volume 59, Issue 6, Pages (June 2001)
DOI: /j x
Copyright © 2001 International Society of Nephrology Terms and Conditions

2. Figure 1
Sepharose CL-4B chromatography of cell surface heparan sulfate proteoglycans (HSPGs). NRK fibroblasts were metabolically labeled with [35S]-sulfate for 24 hours. The medium was removed, and the cell layer was incubated with 20 μg/mL trypsin on ice for five minutes. Portions of (A) the trypsin-released or (B) the trypsin-inaccessible material were applied to DEAE-Sephacel. The bound [35S]-labeled material was eluted, incubated with chondroitin ABC lyase to remove CS/DS chains, and alcohol precipitated. Following reconstitution in 4 mol/L guanidine buffer, the labeled HS material was applied to a column of Sepharose CL-4B. Results are representative of four separate experiments.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

3. Figure 2
Sepharose CL-4B chromatography of cell surface HS-GAG after limited heparitinase digestion. NRK fibroblasts were metabolically labeled with [35S]-sulfate for 24 hours. The medium was removed. The cell layer was incubated for 30 minutes with a cocktail of heparitinases I, II, and III at 37°C, as described, and the released material was applied to a column of Sepharose CL-4B as in Figure 1. Results are representative of three separate experiments.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

4. Figure 3
Immunofluorescent staining of HS-GAG. NRK fibroblasts were grown to confluence in DMEM-Ham's F12 supplemented with 5% FCS. The medium was removed, and the cells were incubated with either (a) buffer alone, (b) heparitinase, or (c) chondroitin ABC lyase at 37°C for 30 minutes. The cells were washed free of enzyme, fixed, and stained for HS-GAG using antibody 10E4. Results are representative of three separate experiments. Magnification ×250.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

5. Figure 4
Cell surface HS-GAGs are required for FGF-2–mediated cell proliferation. Growth-arrested NRK fibroblasts were incubated in buffer alone (□) or with 0.3 U/mL each of heparitinases I, II, and III at 37°C for 30 minutes (▪). The cells were then incubated in serum-free medium containing (a) FGF-2, (b) PDGF-BB, or (c) FCS in the presence or absence of fresh heparitinase for 48 hours. Proliferation was determined by MTT assay. Results are expressed as a mean percentage increase over that of cells incubated in serum-free medium alone ± SEM. *P < 0.05; **P < 0.01, N = 3.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

6. Figure 5
HS-GAGs restore the response of NRK fibroblasts to FGF-2. Cells were grown to subconfluence. The medium was removed, and the cell layer was incubated with 0.3 U/mL heparitinase for 30 minutes. The supernatants were collected, bulked, and heated to 80°C for two hours to inactivate the heparitinases. The HS-GAG–depleted cells were then cultured with 10 ng/mL FGF-2 in the presence of a series of dilutions of the HS-GAG fragments in serum-free medium for 24 hours. Cell numbers were determined using the MTT assay. The results are expressed as the mean percentage ± SEM (N = 4) of the number of cells present after 24 hours in serum-free medium containing only 10 ng/mL FGF-2. *P < 0.05 compared with HS-GAG–depleted cells incubated with FGF-2 alone.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

7. Figure 6
Immunofluorescent staining of sulfate-depleted fibroblasts. Growth-arrested NRK fibroblasts were incubated in sulfate-free DMEM containing 0.5% sulfate-free FCS and (a) 30mmol/L sodium chlorate or (b) 30mmol/L sodium chlorate plus 10mmol/L sodium sulfate for 48 hours. The cells were then fixed and stained for cell surface HS-GAG using antibody 10E4. Results are representative of three separate experiments. Magnification ×250.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

8. Figure 7
Sulfate depletion inhibits the proliferation of renal fibroblasts in response to FGF-2. Growth-arrested NRK fibroblasts were incubated for 48 hours with 10 ng/mL FGF-2 in sulfate-free medium containing sodium chlorate (30 mmol/L) alone or with sodium sulfate (up to 10 mmol/L). Cell numbers were determined using the MTT assay. The results are expressed as the mean ± SEM (N = 6) of the percentage of the number of cells present after 48 hours in medium containing only FGF-2. *P < 0.05; **P < 0.01.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

9. Figure 8
Renal fibroblasts express syndecan mRNA. Total RNA was extracted from NRK 49F cells, and after RT, specific PCR was carried out as described. The individual products were electrophoresed in 3% agarose containing ethidium bromide and the gel photographed under ultraviolet light. Lane (a) base pair ladder markers, (b) syndecan 1, (c) syndecan 2, (d) syndecan 3, (e) syndecan 4, (f) a mixture of each primer product. The gel is representative of five separate experiments.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

10. Figure 9
Basic fibroblast growth factor (FGF-2) induces syndecan 1 mRNA. Growth-arrested NRK fibroblasts were cultured with DMEM-Ham's F-12 supplemented with 10 ng/mL of FGF-2. At the indicated times total RNA was extracted, and RT-PCR amplification was carried out. The products were electrophoresed in 3% agarose containing ethidium bromide, and the gel was photographed under ultraviolet light. α-Actin was used as the housekeeping gene. The gel shown is representative of three separate experiments. The densitometric ratio of syndecan to actin is shown for each time point normalized to the ratio at zero time (mean ± SEM, N = 3).
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions