(A) Western blot showing SMAD2 phosphorylation timecourse upon Activin induction in P19 cells. Cells were treated as indicated and lysates were blotted using the antibodies shown. Arrows indicate the conditions used for the RNA-seq. Below, the phosphorylated SMAD2 (pSMAD2) signal was quantified relative to total SMAD2/3. Untr, untreated (chronic signaling). (B) RNA-seq was performed on P19 cells either untreated or incubated overnight with SB-431542, washed out, then replaced with full media containing SB-431542 for 1 hr (SB-431542 sample), or with full media containing Activin for 1 hr or 8 hr. Shown is a hierarchically-clustered heatmap of log2FC values (relative to SB-431542) for each time point for 747 differentially-expressed genes. (C) log2FC values relative to SB-431542 at each time point plotted for the target genes falling within each of four distinct kinetic clusters. Untr, untreated. (D) Following RNA-seq of control, cycloheximide- or emetine-treated samples in the SB-431542, 1 hr Activin and 8 hr Activin conditions, genes were defined as ‘direct’ or ‘indirect’ depending on whether their pattern changed upon protein synthesis inhibition. Displayed are the relative proportions of direct and indirect target genes in each kinetic cluster. (E) Cells were treated with or without cycloheximide as in (D) and processed for qPCR. Transcript levels for a subset of target genes were quantified relative to Gapdh. Plotted are the means and SEM of two independent experiments performed in duplicate. n.s., not significant. **** corresponds to a p value of < 0.0001; *** corresponds to a p value of < 0.001 and ** corresponds to a p value of < 0.01. (F) Cells were treated overnight with SB-431542, washed out, then stimulated for the indicated times with Activin (blue line) or SB-431542 was added to the Activin-containing media after 1 hr of Activin treatment (red line). qPCR was performed for the genes shown, which are representatives of each of the four distinct transcriptional profiles. Plotted are the means and SEM of three independent experiments performed in duplicate. *** corresponds to a p value of < 0.001 and ** corresponds to a p value of < 0.01.