Fig. 5.

Expression of target genes correlates more strongly with the fold change in nuclear NG-Smad3. (A) To correlate NG-Smad3 dynamics and transcription response within a single cell, we combined live-cell imaging with smFISH. Cells were stimulated with Tgf-β1 and imaged. The same cells were then fixed, stained against specific mRNA, and imaged again. Foci corresponding to individual mRNA molecules were quantified using custom MATLAB scripts. The mRNA transcript counts were then plotted against features of NG-Smad3 response from the same cells. (B) Number of mRNA transcripts plotted against the level (Left) or fold change (Right) of nuclear NG-Smad3 measured in the same cell. The mRNA transcripts were counted after 1 h of Tgf-β stimulation, and plotted here with response features measured at 44 min (snail) and 28 min (ctgf) after ligand stimulation. (C) Plotted is the correlation between mRNA transcripts (at 1 h after ligand stimulation) and NG-Smad3 response measured throughout the entire signaling dynamics. The correlation coefficient is Spearman’s rank correlation coefficient. Error bars are 90% confidence intervals (CI), computed using bootstrap resampling. At each time point after ligand addition, the correlation with fold change (blue) is significantly higher than the correlation with level (orange) (P < 0.01, Steiger’s Z test; a complete statistical analysis is shown in Table S1).