1. Volume 128, Issue 7, Pages 1907-1918 (June 2005)
Oncogenic K-ras Stimulates Wnt Signaling in Colon Cancer Through Inhibition of GSK- 3β 
Jingnan Li, Yusuke Mizukami, Xiaobo Zhang, Won-Seok Jo, Daniel C. Chung 
Gastroenterology 
Volume 128, Issue 7, Pages (June 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
K-rasVal12 can activate the VEGF promoter through TCF-4 binding sites. (A) A 1.9-kilobase VEGF promoter construct containing wild-type or selectively mutated TCF-4 binding sites (TCF-4-c, TCF-4-d) was transiently transfected with phr-GFP or phr-K-ras in Caco-2 cells. The results are expressed as fold induction by phr-K-ras compared with phr-GFP. (B and C) K-rasVal12 regulates Wnt signaling in a PI3-K–dependent manner. Caco-2 and HeLa cells were transiently transfected with pGL3-OT or pGL3-OF reporter constructs and phr-GFP or phr-K-ras. Cells were then treated with 50 μmol/L LY or 20 μmol/L PD98059 for 12 hours before performing luciferase assays. HeLa cells were also treated with 10 μmol/L SB In some experiments (C), the dominant negative PI3-K vector, SRα-P85Δ, was cotransfected. Results are expressed as normalized luciferase activity, defined as the ratio of firefly to Renilla luciferase activity. The data show the mean ± SD of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
K-rasVal12 does not regulate the Wnt pathway through TCF-4. (A) Northern blot analysis showed no change in TCF-4 messenger RNA expression in Caco-2 and 293 cells after transfection of phr-K-ras. (B) Immunoprecipitation and Western blotting analysis failed to show any change in phosphorylation of TCF-4 by phr-K-ras. Total cellular protein was harvested 48 hours after transfection. No β-catenin was coimmunoprecipitated with TCF-4 in 293 cells. pTCF-4, phosphorylated TCF-4.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
K-rasVal12 increases the stability of β-catenin. A total of 100 μg/mL of cycloheximide was added 24 hours after transfection of phr-K-ras or phr-GFP into HeLa cells, and cells were lysed at the indicated time points. β-catenin was detected by Western blotting. Immunoblotting with an actin antibody was performed as a control. The percent of β-catenin remaining at the indicated time points is shown. The β-catenin level was normalized to the actin level at each corresponding time point. Two independent experiments were performed.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
K-rasVal12 increases the levels of nuclear β-catenin but not TCF-4 through a PI3-K-dependent pathway. Western blotting was performed with a β-catenin or TCF-4 antibody using nuclear extracts from Caco-2 cells (15 μg) or HeLa cells (40 μg) transfected with either phr-GFP or phr-K-ras and treated with 50 μmol/L LY or 20 μmol/L PD Immunoblotting with a histone H1 antibody was performed as a control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
K-rasVal12 increases the formation of nuclear β-catenin/TCF-4 complexes. (A) Gel mobility shift assays were performed using nuclear extracts from Caco-2 cells after transient transfection with phr-GFP (lanes 2–5) or phr-K-ras (lanes 6–10). No nuclear extracts were added in lane 1. Sixty-five-fold molar excess of unlabeled wild-type TCF-4 oligo (lane 3) or mutant TCF-4 oligo (lane 4) was added to confirm binding specificity. Antibodies to β-catenin (lane 8), TCF-4 (lane 9), or Cox-2 (lane 10) were added for selected studies. (B) Gel mobility shift assays were performed with nuclear extracts prepared from HeLa cells transiently transfected with phr-GFP (lanes 2 and 4) or phr-K-ras (lanes 3 and 5–12). Cells were pretreated with 10 μmol/L SB for 12 hours (lanes 4–12). No nuclear extracts were added in lane 1. Fifty-fold molar excess of unlabeled wild-type TCF-4 oligo (lane 7) or mutant TCF-4 oligo (lane 8) was added to confirm binding specificity. β-catenin (lane 10), TCF-4 (lane 11), or Cox-2 (lane 12) antibody was added for selected experiments. (C) After transfection of phr-GFP (lanes 1 and 5) or phr-K-ras (lanes 2–4 and 6–8) in Caco-2 cells and HeLa cells, 50 μmol/L LY (lanes 3 and 7) or 20 μmol/L PD98059 (lanes 4 and 8) was added for 8 hours before harvesting nuclear protein. A total of 10 μmol/L SB was also added to HeLa cells (lanes 5–8). Three independent experiments were performed.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
K-rasVal12 does not regulate the binding of GSK-3β to Axin. In Caco-2 and HeLa cells, Flag-Axin was cotransfected with phr-K-ras or phr-GFP. Twelve hours before harvesting protein, 50 μmol/L LY was added. Extracts were immunoprecipitated with a Flag antibody and immunoblotted with a Flag and GSK-3β antibody.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
K-rasVal12 decreases GSK-3β activity independent of phosphorylation at serine 9. (A) phr-K-ras or phr-GFP was transfected into Caco-2 and HeLa cells, and then 50 μmol/L LY was added 12 hours before harvesting protein. Western blotting and GSK-3β assays were performed as described in Materials and Methods. Kinase activity was expressed as the percentage of control activity with empty vector transfection. (B) pGL3-OT or pGL3-OF reporter constructs were transiently transfected with phr-GFP or phr-K-ras and HA-GSK-3β or HA-GSK-3βAla9. Results are expressed as normalized luciferase activity. Cell lysates were immunoprecipitated with HA-tag antibody and then assayed for GSK-3β activity. (C) phr-K-ras or phr-GFP was cotransfected with Flag-Axin. Cell lysates were immunoprecipitated with Flag antibody. These extracts were then used for either immunoblotting or GSK-3β activity assays. The kinase activity is shown as the mean ± SD of 3 independent experiments. (D) HeLa cells were transfected with either phr-K-ras or phr-GFP. A total of 10 μmol/L SB21673 was then added to selected wells for 12 hours, and cellular lysates were assayed for GSK-3β kinase activity as described in Materials and Methods. Results are presented as the mean ± SD of 2 independent experiments performed in duplicate. GSK-3β kinase activity in HeLa cells transfected with phr-GFP has been normalized to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions