Effect of Smad7 on erythroid differentiation induced by activated forms of type I activin receptors.
(A) Promotion of erythroid differentiation of F5-5 cells by expression of constitutively activated ActR-I (QD) or ActR-IB (TD). Cells were infected with various combinations of vector viruses expressing ActR-I (QD), ActR-IB (TD), and ActR-II. Infected cells were supplemented with medium to achieve the initial density of 3 × 104cells/mL and incubated for a week. At the end of the incubation period, a portion of the culture was examined by using benzidine staining for hemoglobin and the rest were collected for RNA extraction. The results shown are the percentages of total cells that contained hemoglobin. Values are the mean and SD from triplicate assays. (B) Total RNA harvested from the above cells was analyzed with RT-PCR for the presence of β-globin mRNA as a marker of erythroid differentiation. G3PDH served as a loading control. (C and D) Smad7 efficiently inhibits erythroid differentiation induced by ActR-I (QD) but only inefficiently inhibits that induced by ActR-IB (TD). F5-5 cells were infected with puromycin-selectable virus expressing Smad7 or with control virus. Stably transduced cells, F5-5 (Smad7) cells and F5-5 (control) cells, were selected by incubation in the presence of 1 μg/mL puromycin for 1 week. The puromycin-resistant cells were infected with combinations of ActR-I (QD), ActR-IB (TD), and ActR-II viruses, as indicated. After another week of culture, a portion of the culture was used for benzidine staining and the rest were used for RT-PCR analysis of β-globin gene expression.
