Figure 2

Smad7 sustains CRC cell proliferation and survival. (a) Total proteins extracted from two CRC cell lines (i.e., DLD-1 and HCT-116) and from normal colonic epithelial cells (CEC) were evaluated for Smad7 expression by western blotting. β-actin was used as loading control. One of two representative experiments in which similar results were obtained is shown (b) Smad7 antisense oligonucleotide (AS) downregulates Smad7 protein expression in HCT-116 cells. HCT-116 cells were transfected with either Smad7 sense oligonucleotide (S) (2 μg/ml) or increasing doses (0.5–2 μg/ml) of Smad7 AS. Smad7 and β-actin were analyzed by western blotting. One of three representative experiments is shown. (c) Smad7 AS inhibits the growth of CRC cell lines. HCT-116 and DLD-1 cells were either left untreated (Untr) or transfected with Smad7 AS or Smad7 S. The percentage of proliferating cells was evaluated by flow cytometry. Data indicate mean±S.D. of four experiments (HCT-116: Smad7 S-transfected cells versus Smad7 AS-transfected cells, *P<0.001; DLD-1: Smad7 S-transfected cells versus Smad7 AS-transfected cells, ^†P<0.001). Right insets. Representative histograms of HCT-116 cells transfected with SMAD7 S and AS. Numbers above lines indicate the percentages of proliferating cells. (d) Smad7 AS induces HCT-116 cells to arrest in S phase of cell cycle. Cells were either left untreated (Untr) or transfected with Smad7 S or AS. After 24 h cells were washed with PBS and cultured for further 24 h. Cell cycle distribution was assessed by flow cytometry. Values are the percentages of cells in the different phases of cell cycle and indicate mean±S.D. of four experiments. A significant increase in the number of cells that accumulate in S phase (*P<0.001) and a significant decrease in the number of cells in G0/G1 (**P<0.01) was seen in Smad7 AS-transfected cells as compared with Smad7 S-transfected cells. (e) Smad7 AS enhances the expression of p-CDK2. HCT-116 cells were either left untreated (Untr) or transfected with Smad7 AS or S for 24 h. p-CDK2, CDK2 and β-actin expression was analyzed by Western blotting after further 24 h of culture. One of three representative experiments in which similar results were obtained is shown. (f) Inhibition of Smad7 with AS induces HCT-116 cell death at late time points. Cells were either left untreated (Untr) or transfected with Smad7 S or AS (1 μg/ml). After 24 h cells were washed with PBS and cultured for further 24 (left panel) or 48 (middle panel) hours. Data indicate the percentage of cell death as assessed by flow cytometry analysis of Annexin V (AV) and/or propidium iodide (PI)-positive cells and are expressed as mean±S.D. of three experiments. Right panel. Representative dot-plots showing the percentages of AV- and/or PI-positive cells after 48 h