Additional Supporting Information may be found in the online version of this article.

IJC_26257_sm_suppfig1.ppt141KSupporting Figure 1 Ligand-dependent GCC signaling induces rapid VASP Ser phosphorylation in colon cancer cells. (a) VASP-specific immunoblots of representative experiments (repeated 3 times) analyzing total cell lysates from various human colon carcinomas. GAPDH, the loading control. (b) Immunoblots of representative time-course experiments with T84 human colon carcinoma cells treated with the GCC agonist (1 μM). pS157-VASP and pS239-VASP, VASP phosphorylated at Ser157 and Ser239, respectively. Villin, the loading control.
IJC_26257_sm_suppfig2.ppt128KSupporting Figure 2 GCC signaling trough PKG induces VASP Ser phosphorylation in colon cancer cells. Immunoblots of representative experiments with HCT116 (a) and T84 (b) human colon carcinoma cells. Treatments were: the cGMP analog 8-br-cGMP (5 mM, 30 min); the GCC ligand ST (1 μM, 5 min), and the selective PKG inhibitor DT2 (5 μM, 30 min; ref. 17 in manuscript). pS157-VASP and pS239-VASP, VASP phosphorylated at Ser157 and Ser239, respectively; GAPDH, the loading control.
IJC_26257_sm_suppfig3.ppt1497KSupporting Figure 3 GCC signaling through VASP Ser phosphorylation is dysregulated in colon cancer. IHC images (magnification, 20x) of serial sections from primary tumor (Tumor) and matched normal adjacent tissue (NAT) of a patient with colorectal cancer. Tissues were stained with the specific primary antibody (brown) and hematoxylin (blue, nuclei). A rabbit primary antibody (1:600 dilution) from Sigma-Aldrich was employed for VASP phosphorylated at Ser239. pS157-VASP and pS239-VASP, VASP phosphorylated at Ser157 and Ser239, respectively.
IJC_26257_sm_suppfig4.ppt316KSupporting Figure 4. Colon cancer cells with VASP phosphomutants are resistant to GCC-mediated phosphorylation. Plasmid constructions and cell transductions were performed as detailed in Materials and Methods. (a) Representative immunoblots of total VASP (VASP) and the loading control (GAPDH) from T84 cells stably expressing the indicated MSCV-driven genes, including VASP-S157A (S157A), VASP-S239A (S239A) and VASP-AA (AA). Wild-type (WT) T84 cells, and cells expressing GFP or VASP represent the control conditions. (b) Representative immunoblots and associated graphs (reflecting mean ± SEM of 3 independent experiments) of VASP phosphorylated at Ser157 (pS157-VASP; left) and VASP phosphorylated at Ser239 (pS239-VASP; right) in T84 cells (genetically manipulated as in a) treated for 5 min with ST (1 μM). Values in graphs are relative levels normalized to respective loading (villin) and vehicle (PBS) controls. *, P < 0.05, versus respective PBS control.
IJC_26257_sm_suppfig5.ppt87KSupporting Figure 5 GCC signaling induces rapid VASP removal from colon cancer cell filopodia. Exponential decay curve fitted to the data presented in Fig. 5d, examining the effects of the GCC agonist ST (1 μM) on VASP localization at filopodia. Dotted lines are 95% confidence limits. The curve was generated employing the software Prism 5.0 (GraphPad).
IJC_26257_sm_supptable1.doc46KSupporting Table 1.

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