(a,b) Boxplots showing the maximal MS2-GFP intensity levels reached on actively transcribing CCND1-MS2 genes during 6 hr in Wnt3a-treated and mock-treated (Con) cells, when (a) the gene was either not transcribing before the addition of Wnt3a or mock-treatment (‘off’, n(Wnt3a) = 27, n(Con) = 22, p=0.0001) or (b) if the gene was already active (‘on’, n(Wnt3a) = 37, n(Con) = 52, p=0.0006). The median is indicated by a red line, the box represents the interquartile range, the whiskers represent the maximum and minimum values, and red dots represent outliers. (c,d) Boxplots showing the time required to reach the maximal intensity levels when (c) the gene was either not transcribing before the addition of Wnt3a (‘off’, p=0.03) or (d) if the gene was already active (‘on’, p=0.42). (e) YFP-β-catenin (yellow) together with RNA FISH images obtained with a probe hybridizing to the MS2 region in the 3’UTR of the CCND1-MS2 mRNA (cyan), showing CCND1 nascent mRNAs on active genes (large dots) and cellular mRNAs (small dots) in Wnt3a-treated cells (2 hr), in comparison to YFP-β-catenin levels. Nuclei are stained with Hoechst (pseudo-colored red). Bottom row is the pseudo-colored YFP signal using the ImageJ ‘Royal’ look-up table. Cells are numbered. Bar = 10 μm. (f) Quantification of the number of cellular CCND1-MS2 mRNAs (ordered from low to high) compared to YFP-β-catenin levels. (g) Quantification of the number of nascent CCND1-MS2 mRNAs compared to YFP-β-catenin levels. (h,i) Correlation analysis between (h) the number of cellular CCND1-MS2 mRNAs and YFP-β-catenin levels and (i) between the number of nascent CCND1-MS2 mRNAs and YFP-β-catenin levels. Blue dots – subpopulation with low nuclear YFP-β-catenin levels and low numbers of cellular/nascent CCND1-MS2 mRNAs. Red dots – subpopulation with high nuclear YFP-β-catenin levels and high numbers of cellular/nascent CCND1-MS2 mRNAs. Total correlation score between the number of cellular/nascent CCND1-MS2 mRNAs and YFP-β-catenin levels is 0.88 and 0.59, respectively. (j) The field from panel e demonstrating higher intensity of active CCND1-MS2 genes in cells with high nuclear YFP-β-catenin levels (red arrows) compared to cells with low nuclear YFP-β-catenin levels (yellow arrows). Active genes are pseudo-colored using the ImageJ ‘Red Hot’ look-up table. The fluorescent signal of the active genes was enhanced using ImageJ 'Spot Enhancing Filter 2D'. This enhancement led to the reduced detectability of single mRNAs in this presentation of the image, in order to emphasize the difference in transcriptional activity between low and high levels of nuclear YFP-β-catenin. Bar = 10 μm.