Figures & data
Table 1. The primers used in this study.
Figure 1. Radiation induced DNA damage and inhibited cell proliferation in CNE-2 cells. (A) γH2AX signal presented the DNA damage was increased together with the increased doses of radiation. (B) the cell viability was significantly decreased followed with the increased dose usage of radiation. (C, D), the amounts of the cell colonies were significantly decreased followed with the increased doses of radiation. * P < 0.05 vs. 0 Gy; ** P < 0.01 vs. 0 Gy. Scale bar: 10 μm.
Figure 2. Radiation induced the cell cycle arrest and promoted cell apoptosis in CNE-2 cells. (A) radiation increased the percentage of the cells in G2/M phase. (B) the cell apoptosis and necrosis were enhanced after radiation. * P < 0.05 vs. 0 Gy; ** P < 0.01 vs. 0 Gy.
Figure 3. Western blotting analysis. Radiation activated the ATM pathway, increased the expression of Smad7 and inhibited the phosphorylation of Smad3.
Figure 4. Application of TGF-β1 increased the sensitivity of CNE-2 cells to radiation. (A) the DNA damage was enhanced by TGF-β1, compared with 0 Gy and 6 Gy group. (B) TGF-β1 treatment significantly decreased of the cell viability of CNE-2 cells compared with the 0 Gy and 6 Gy radiation groups. (C, D) the amounts of the cell colonies were significantly decreased after the TGF-β1 treatment.** P < 0.01 vs. 0 Gy.
Figure 5. The effect to the cell cycle and cell apoptosis of CNE-2 cells after TGF-β1 treatment. (A) the percentage of the cells was increased in G2/M phase. (B) the cell apoptosis and necrosis were significantly enhanced. ** P < 0.01 vs. 0 Gy; # P < 0.05 vs. 6 Gy.
Figure 6. TGF-β1 activated the ATM pathway, increased the expression of Smad7 and inhibited the phosphorylation of Smad3.
Figure 7. Overexpression of Smad3 or suppression of Smad7 decreased the damage to CNE-2 cells after radiation. (A) the Smad3 overexpression vector and Smad7 suppression shRNA was transfected to CNE-2 cells successfully. (B) Smad3 mRNA expression was significantly increased in the Smad3 group but significantly decreased in the shSmad7 group. ** P < 0.01 vs. NC group. (C) compared with the NC+6 Gy group, the DNA damage was decreased in Smad3+6 Gy group and shSmad7+6 Gy group.
Figure 8. Overexpression of Smad3 or suppression of Smad7 affected the cell proliferation and colony formation. (A) the cell viability was slightly increased in Smad3+6 Gy group and shSmad7+6 Gy group. * P < 0.05 vs. NC group; #P < 0.05 vs.Smad3 group; & P < 0.05 vs. shSmad7 group. (B) overexpression of Smad3 or suppression of Smad7 increased the cell colony formation; ** P < 0.01 vs. NC group; # P < 0.05 vs. NC+6 Gy group.
Figure 9. Overexpression of Smad3 or suppression of Smad7 affected the cell cycle and cell apoptosis. (A) the cells in G2/M phase were decreased, while the cells in G1 and S phase were increased. (B) Cell apoptosis and necrosis were significantly decreased. Compared Smad3 group, the cell apoptosis was increased in Smad3+6 Gy group; Compared with shSmad7 group, the cell apoptosis was increased in shSmad7+6 Gy group. * P < 0.05, ** P < 0.01 vs. NC group; #P < 0.05 vs. NC+6 Gy group.
Figure 10. Overexpression of Smad3 or suppression of Smad7 attenuated the activation of ATM pathway.
Figure 11. Overexpression of Smad3 or suppression of Smad7 suppressed the activation of ATM pathway, and decreased the sensitivity of tumor to radiation in nude mice. (A, B), compared with CNE-2+6 Gy group, the volume and the weight of the tumor was increased obviously in Smad3+6 Gy group and shSmad7+6 Gy group. (C) the mRNA level of Smad3 and Smad7 was detected by RT-PCR. * P < 0.05,** P < 0.01.
Figure 12. Immunohistochemical staining in evaluating the robustness of ATM pathway and Smad3/7 cascade before and after treated with radiation.
Figure 13. Western blotting to determine the expression level of cleaved caspase3 in formed tumors.
