Supplementary Data

Supplementary Figure 1. Effects of various kinds of protein kinase inhibitors on the mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3UTR and pcDNA6/V5-TTP. At 24 h post-transfection, cells were treated with PKC (Ro-32-0432), phosphatidylinositol 3-kinase (PI3K) (LY294002), mTOR (Rapamycin), JNK (JNK inhibitor I), or MEK1 (PD98059) inhibitors at indicated concentration for 12 h, and luciferase activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from psiCHECK2-VEGF 3UTR-transfected cells was set to 1. Each bar represents the mean S.D. of three independent experiments (*p<0.05, **<0.01, ***p<0.001). ns, not significant.

Supplementary Figure 2. CK2 inhibitor TBB decreases the mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3UTR and pcDNA6/V5-TTP. At 24 h posttransfection, cells were treated with CK2 inhibitor TBB at indicated concentrations for 12 h, and luciferase activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from psiCHECK2-VEGF 3UTR-transfected cells was set to 1. Each bar represents the mean S.D. of three independent experiments (*p<0.05, **<0.01, ***p<0.001). ns, not significant.

Supplementary Figure 3. Both kinase inhibitors DRB and SB 203580 do not affect the mRNA decaying activity of TTP and the expression level of VEGF mRNA. (A) Colo320 cells were transfected with psiCHECK2-VEGF 3UTR. At 24 h post-transfection, cells were treated with CK2 inhibitor DRB or p38 MAPK inhibitor SB203580 at indicated concentrations for 12 h, and luciferase activity was determined. Renilla luciferase activity
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was normalized to firefly activity. The luciferase activity obtained from psiCHECK2-VEGF 3UTR-transfected cells was set to 1. Each bar represents the mean S.D. of three independent experiments. ns, not significant. (B) Colo320 cells were treated with CK2 inhibitor DRB or p38 MAPK inhibitor SB203580 at indicated concentrations for 12 h and VEGF mRNA was determined by quantitative real-time PCR. The expression level obtained from no treated cells was set to 1. Each bar represents the mean S.D. of three independent experiments. ns, not significant.

Supplementary Figure 4. CK2 inhibitor DRB increases the phosphorylation of TTP. Colo320 cells were transfected with pcDNA6/V5-TTP, and at 24 h post-transfection cells were treated with DRB in the presence or absence of MG132 at the indicated concentrations. TTP protein levels were determined by immunoblotting with anti-V5 antibody. The arrowhead and arrow indicate unphosphorylated and phosphorylated forms of TTP, respectively.

Supplementary Figure 5. Inhibition of CK2 by TBB decreases the expression of MKP-1. Colo320 cells transfected with pcDNA6/V5-TTP were treated with TBB and cells were harvested at 12 h after TBB treatment. Cell lysates were analyzed for TTP and MKP-1 by immunoblotting.

Supplementary Figure 6. Proteasome inhibitor MG132 abolishes the decrease of MKP-1 induced by DRB. Colo320 cells were treated with DRB in the presence or absence of MG132. The expression level of MKP-1 was determined by immunoblotting with anti-MKP1 antibody.

Supplementary Figure 7. TGF- does not affect the TTP promoter activity. Colo320 cells
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were transfected with pGL3/TTP promoter. At 24 h post-transfection, cells were treated with 2 ng/ml of TGF- in the presence or absence of DRB for 6 h and luciferase activity was determined. The luciferase activity obtained from untreated cells was set to 1. Each bar represents the mean S.D. of three independent experiments.

Supplementary Figure 8. Inhibition of CK2 by CK2 inhibitor DRB decreases the expression level of 14-3-3. Colo320 cells were treated with DRB. The expression level of 14-3-3 was determined by immunoblotting with anti-14-3-3 antibody.

Supplementary Figure 9. TGF- slightly increases DAB2 expression. Colo320 cells were treated with 2 ng/ml of TGF- for 4 h. The expression level of DAB2 was determined by semi-quantitative PCR using PCR primers: 5-CCAACAGAAAGCAAAGATATCC-3, 5GTTGGTCGAGGAAGAGAAC-3.

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