Inhibitory effect of Smad7 on erythroid differentiation induced by activin.

(A) Hemoglobin accumulation in F5-5 mouse erythroid leukemia cells and in derivatives. F5-5 cells were infected with control or Smad7 transducing virus and selected for acquiring G418 resistance. After 5 days of culture with (+) or without (−) 1 nmol/L activin, cells were stained with benzidine to reveal hemoglobin content. F5-5 (control) indicates G418-resistant control cells, and F5-5 (Smad7) indicates Smad7 virus-transduced cells. (B) Immunoblotting analysis of Smad7 stably expressed in clonal cells. FS7-5 and FS7-15 cells were cloned from single G418-resistant cells that had been infected with Smad7 expression vector. The whole-cell lysate of 2 × 10⁴ cells (left) or the indicated amount (0.4, 2, or 10 ng) of the bacterially expressed, purified Smad7 protein (amino acids 91-426; right) was loaded into each lane and subjected to electrophoresis and blotting. Smad7 was detected with specific antibody against Smad7. Densitometric analysis indicated that 2 × 10⁴ F5-5 parent cells, FS7-5 clonal cells, and FS7-15 clonal cells contained less than 4 pg, 100 pg, and 20 pg, respectively, of Smad7 protein. (C) Effect of Smad7 level on activin-induced hemoglobin synthesis. F5-5, FS7-5, and FS7-15 cells were cultured for 5 days in the absence (−) or presence (+) of 1 nmol/L activin and assessed for hemoglobin accumulation. The results shown are the percentages of total cells that contained hemoglobin. Values are the mean and SD from 3 separate experiments.