Smad3 mediates tumor suppression by TGF-β in the MCF10A model of breast cancer progression. (A) Schematic illustration of the MCF10A-based xenograft model of breast cancer progression. TGF-β has tumor-suppressor activity in M3 cells but this effect is lost in M4 cells and instead TGF-β promotes metastasis. (B) Knockdown of Smad2 and Smad3 protein in M3 and M4 cells was verified by Western blot, quantitated relative to the β-actin loading control and normalized relative to the shGFP condition for each cell line. (C) Relative contributions of Smad2 and Smad3 to the tumor-suppressive effect of TGF-β. Mice were orthotopically implanted with M3 cells or M4 cells, genetically modified to stably express shSmad2, shSmad3 or the control shGFP, and tumor volumes were assessed after seven weeks (M3) or four weeks (M4). Bars indicate median +/−interquartile range; P <0.05 was statistically significant, Mann-Whitney U test. ns, not significant. (D) Kinetics of Smad3 phosphorylation. Western blot of total Smad3 protein and C-terminal phosphorylated Smad3 (Smad3-CP) levels at various time points after TGF-β treatment in M1 to 4 cells. Total Smad3 is shown for t = 0 h. (E) Western blot showing linker phosphorylated Smad3 (Smad3-LP) at 1 hour after treatment with 2 ng/ml TGF-β. TGF-β, transforming growth factor beta.