Figure 5

Transcriptional suppression of Smads by 3′-deoxyadenosine (3′-DA). (a) NRK-52E cells were treated with 3′-DA for indicated time periods and subjected to northern blot analysis for Smad mRNAs. The levels of Smad1, Smad2 and Smad3 mRNAs were normalized by the level of GAPDH, and the kinetics is shown in the graph. (b) Cells were treated with 3′-DA for indicated concentrations for 9 h and subjected to northern blot analysis of Smads. (c) Cells were treated with 3′-DA in the presence of DPSPX, MRS1523 or NBTI for 9 h and subjected to northern blot analysis. Quantification of individual signals is shown in graphs. (d) SM43 cells were treated with 3′-DA for 9 h and subjected to northern blot analysis. Quantification of individual signals is shown in the graph. (e) NRK-52E cells were pretreated with or without 3′-DA for 30 min, exposed to actinomycin D (Act D; 5 μg/ml) for 0–6 h and subjected to northern blot analysis. The levels of Smad1, Smad2 and Smad3 mRNAs were normalized by the level of GAPDH, and relative intensity of Smad mRNAs is shown in graphs. (f) Cells were transiently transfected with pGL2-F-1005/+93Smad1, treated with or without 3′-DA for 24 h and subjected to chemiluminescent assay. Asterisks indicate statistically significant differences. NS, not statistically significant; RLU, relative light unit.