1. Suppression of fibroblast growth factor receptor signaling inhibits pancreatic cancer growth in vitro and in vivo 
Markus Wagner, Martha E. Lopez, Mitch Cahn, Murray Korc 
Gastroenterology 
Volume 114, Issue 4, Pages (April 1998)
DOI: /S (98)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Generation of a truncated FGFR-1 expression vector. The extracellular domain from a 1.2-kb BamHI fragment of pCD115 FGFR-1 cDNA was ligated into the 3Zf plasmid, followed by ligation with a PCR-generated (from human placenta cDNA) 315-bp cDNA fragment corresponding to nucleotides 901–1215 of human FGFR-1 cDNA. The resulting cDNA, designated FGFR405, encoded a protein that consisted of the extracellular and transmembrane (TM) domains of FGFR-1 and that terminated at residue 405 of the full-length receptor, yielding a receptor that is devoid of the cytoplasmic domain. Authenticity was confirmed by sequencing.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Analysis of FGFR-1 expression. (A) Northern blot analysis. Total RNA (20 μg/lane) was extracted from parental PANC-1 cells, cells transfected with pRK5 only, and cells transfected with pRK-FGFR405. A 32P-labeled FGFR-1 cDNA probe (1,000,000 counts per minute [cpm]/mL; 2-day exposure) was used for hybridizations. A 7S cDNA probe (50,000 cpm/mL; 12-hour exposure) was used to confirm equivalent loading of lanes. The positions of the 28S and 18S ribosomal RNA are indicated on the right. Numbers above the lanes represent individual cell clones. (B) Western blot analysis. Cell lysates were immunoprecipitated with anti–FGFR-1 antibodies, electrophoresed by 6% SDS-PAGE, and transferred to PVDF membranes. Blots were probed with anti–FGFR-1 antibodies and subjected to chemiluminescence Western blotting.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

4. Fig. 2
Analysis of FGFR-1 expression. (A) Northern blot analysis. Total RNA (20 μg/lane) was extracted from parental PANC-1 cells, cells transfected with pRK5 only, and cells transfected with pRK-FGFR405. A 32P-labeled FGFR-1 cDNA probe (1,000,000 counts per minute [cpm]/mL; 2-day exposure) was used for hybridizations. A 7S cDNA probe (50,000 cpm/mL; 12-hour exposure) was used to confirm equivalent loading of lanes. The positions of the 28S and 18S ribosomal RNA are indicated on the right. Numbers above the lanes represent individual cell clones. (B) Western blot analysis. Cell lysates were immunoprecipitated with anti–FGFR-1 antibodies, electrophoresed by 6% SDS-PAGE, and transferred to PVDF membranes. Blots were probed with anti–FGFR-1 antibodies and subjected to chemiluminescence Western blotting.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

5. Fig. 3
Phosphorylation of FGFR-1. Parental PANC-1 cells and clones 15, 28, and 51 transfected with pRK-FGFR405 were incubated in the presence or absence of 1 nmol/L bFGF as indicated. After cell lysis, FGFR-1 was immunoprecipitated with anti–FGFR-1 antibody. Immunoprecipitates were then electrophoresed by 6% SDS-PAGE and transferred to PVDF membranes. The membranes were probed with antiphosphotyrosine antibodies and subjected to chemiluminescence Western blotting.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

6. Fig. 4
Analysis of MAP-kinase activation. (A) Effects of bFGF and TGF-α on MAP-kinase activation in PANC-1 cells and transfected clones 28 and 51. Subconfluent parental PANC-1 cells or clones 28 and 51 expressing FGFR405 were incubated in the absence (−) or presence of 1 nmol/L bFGF (b, 40 μg protein/lane) or TGF-α (T, 10 μg protein/lane) for 5 minutes, lysed, electrophoresed by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with an anti-active MAP kinase antibody and subjected to chemiluminescence Western blotting. Membranes were then stripped and reprobed with an anti–ERK-2 antibody to confirm equivalent loading of lanes. (B) Comparison of the actions of bFGF, aFGF, FGF-4, and TGF-α in PANC-1 cells and clone 15. Subconfluent PANC-1 cells or clone 15 cells expressing FGFR405 were incubated as above but in the absence (−) or presence of 1 nmol/L aFGF (a), bFGF (b), FGF-4 (4), or TGF-α (T). Protein lysates were subjected to immunoblotting as above with an anti-active MAP kinase antibody. Membranes were then stripped and reprobed with an anti–ERK-2 antibody. (C) Comparison of the actions of bFGF and aFGF in MIA PaCa-2 cells and transfected MIA PaCa-2 clone 35. Subconfluent cells were incubated as above but in the absence (−) or presence of 1 nmol/L aFGF (a) or bFGF (b). Protein lysates were subjected to immunoblotting as above with an anti-active MAP kinase antibody. Membranes were then stripped and reprobed with an anti–ERK-2 antibody.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

7. Fig. 4
Analysis of MAP-kinase activation. (A) Effects of bFGF and TGF-α on MAP-kinase activation in PANC-1 cells and transfected clones 28 and 51. Subconfluent parental PANC-1 cells or clones 28 and 51 expressing FGFR405 were incubated in the absence (−) or presence of 1 nmol/L bFGF (b, 40 μg protein/lane) or TGF-α (T, 10 μg protein/lane) for 5 minutes, lysed, electrophoresed by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with an anti-active MAP kinase antibody and subjected to chemiluminescence Western blotting. Membranes were then stripped and reprobed with an anti–ERK-2 antibody to confirm equivalent loading of lanes. (B) Comparison of the actions of bFGF, aFGF, FGF-4, and TGF-α in PANC-1 cells and clone 15. Subconfluent PANC-1 cells or clone 15 cells expressing FGFR405 were incubated as above but in the absence (−) or presence of 1 nmol/L aFGF (a), bFGF (b), FGF-4 (4), or TGF-α (T). Protein lysates were subjected to immunoblotting as above with an anti-active MAP kinase antibody. Membranes were then stripped and reprobed with an anti–ERK-2 antibody. (C) Comparison of the actions of bFGF and aFGF in MIA PaCa-2 cells and transfected MIA PaCa-2 clone 35. Subconfluent cells were incubated as above but in the absence (−) or presence of 1 nmol/L aFGF (a) or bFGF (b). Protein lysates were subjected to immunoblotting as above with an anti-active MAP kinase antibody. Membranes were then stripped and reprobed with an anti–ERK-2 antibody.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

8. Fig. 4
Analysis of MAP-kinase activation. (A) Effects of bFGF and TGF-α on MAP-kinase activation in PANC-1 cells and transfected clones 28 and 51. Subconfluent parental PANC-1 cells or clones 28 and 51 expressing FGFR405 were incubated in the absence (−) or presence of 1 nmol/L bFGF (b, 40 μg protein/lane) or TGF-α (T, 10 μg protein/lane) for 5 minutes, lysed, electrophoresed by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with an anti-active MAP kinase antibody and subjected to chemiluminescence Western blotting. Membranes were then stripped and reprobed with an anti–ERK-2 antibody to confirm equivalent loading of lanes. (B) Comparison of the actions of bFGF, aFGF, FGF-4, and TGF-α in PANC-1 cells and clone 15. Subconfluent PANC-1 cells or clone 15 cells expressing FGFR405 were incubated as above but in the absence (−) or presence of 1 nmol/L aFGF (a), bFGF (b), FGF-4 (4), or TGF-α (T). Protein lysates were subjected to immunoblotting as above with an anti-active MAP kinase antibody. Membranes were then stripped and reprobed with an anti–ERK-2 antibody. (C) Comparison of the actions of bFGF and aFGF in MIA PaCa-2 cells and transfected MIA PaCa-2 clone 35. Subconfluent cells were incubated as above but in the absence (−) or presence of 1 nmol/L aFGF (a) or bFGF (b). Protein lysates were subjected to immunoblotting as above with an anti-active MAP kinase antibody. Membranes were then stripped and reprobed with an anti–ERK-2 antibody.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

9. Fig. 5
Effects of bFGF on cell growth. (A) Anchorage-dependent growth. Wild-type cells (PANC-1), cells transfected with pRK5 only (P-pRK5), and clones 15, 28, and 51 were plated at 3 × 103 cells/well in 96-well plates with 0.2 mL of normal growth medium. Medium was replaced every second day, and the relative cell numbers were determined by using the MTT assay. Values represent means ± SD of 10 determinations. (B) Anchorage-independent growth. Parental PANC-1 cells, PANC-1 cells transfected with pRK5 only (P-pRK5), and transfected clones were incubated in the presence or absence of bFGF. Colonies with a diameter of >80 μm were counted. Each point is the mean ± SE of triplicate determinations from three different sets of experiments (n = 9). *P < 0.05, **P < vs. parental PANC-1 cells in the absence of bFGF.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

10. Fig. 5
Effects of bFGF on cell growth. (A) Anchorage-dependent growth. Wild-type cells (PANC-1), cells transfected with pRK5 only (P-pRK5), and clones 15, 28, and 51 were plated at 3 × 103 cells/well in 96-well plates with 0.2 mL of normal growth medium. Medium was replaced every second day, and the relative cell numbers were determined by using the MTT assay. Values represent means ± SD of 10 determinations. (B) Anchorage-independent growth. Parental PANC-1 cells, PANC-1 cells transfected with pRK5 only (P-pRK5), and transfected clones were incubated in the presence or absence of bFGF. Colonies with a diameter of >80 μm were counted. Each point is the mean ± SE of triplicate determinations from three different sets of experiments (n = 9). *P < 0.05, **P < vs. parental PANC-1 cells in the absence of bFGF.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

11. Fig. 6
Xenograft formation. (A and B) Exponentially growing cells (1 × 106) were inoculated subcutaneously in athymic nude mice and allowed to grow for 6 weeks. Solid arrows indicate sites of injection of parental or sham-transfected PANC-1 cells; open arrows indicate sites of injection of PANC-1 cells transfected with pRK-FGFR405. (C) Tumors were measured externally on indicated days in two dimensions, and tumor volume was determined by the equation vol = (l × w2) × 0.5, where vol is volume, l is length, and w is width of the tumor. Values are means ± SE (n=4).
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

12. Fig. 6
Xenograft formation. (A and B) Exponentially growing cells (1 × 106) were inoculated subcutaneously in athymic nude mice and allowed to grow for 6 weeks. Solid arrows indicate sites of injection of parental or sham-transfected PANC-1 cells; open arrows indicate sites of injection of PANC-1 cells transfected with pRK-FGFR405. (C) Tumors were measured externally on indicated days in two dimensions, and tumor volume was determined by the equation vol = (l × w2) × 0.5, where vol is volume, l is length, and w is width of the tumor. Values are means ± SE (n=4).
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

13. Fig. 6
Xenograft formation. (A and B) Exponentially growing cells (1 × 106) were inoculated subcutaneously in athymic nude mice and allowed to grow for 6 weeks. Solid arrows indicate sites of injection of parental or sham-transfected PANC-1 cells; open arrows indicate sites of injection of PANC-1 cells transfected with pRK-FGFR405. (C) Tumors were measured externally on indicated days in two dimensions, and tumor volume was determined by the equation vol = (l × w2) × 0.5, where vol is volume, l is length, and w is width of the tumor. Values are means ± SE (n=4).
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions