In response to pheromone treatment, the number, intensity, and distribution of MFA2 mRNA granules increases, whereas PGK1 mRNA granules remain unchanged. (A) The WT strain coexpressing U1A-GFP and either MFA2-U1A (ySA20) or PGK1-U1A (ySA32) was grown to the early exponential phase and treated with 3 nM αF for 60 min, as indicated. Bar, 5 µm. (B) Quantification of the number of MFA2 or PGK1 mRNA granules in treated and untreated cells 1 h after adding αF. Histograms illustrating the distribution of mRNA granules per cell are shown. Error bars represent the standard deviation of three independent experiments. n = 200–350 cells. Averaging these numbers revealed that, in response to αF, the number of MFA2 mRNA–containing granules per cell increased twofold, whereas their intensity increased threefold. No change in the number and intensity of the PGK1 mRNA–containing granules was observed in response to a similar treatment with αF. (C) Intensity histograms of MFA2 and PGK1 mRNA granules in αF-treated cells. The WT strain coexpressing U1A-GFP and either MFA2-U1A (ySA20) or PGK1-U1A (ySA32) was treated with αF for 2 h. Images were captured, threshold, and analyzed for Integrated Optical Density (IOD) using the ImageJ program. IOD distributions are depicted as histograms (see Materials and methods). 200–350 cells displaying a clear shmoo were analyzed. Distribution of intensities of the MFA2 and PGK1 mRNA granules in a whole cell (I and II) and in the shmoo region (III-IV) are shown. Note the clear distinction between the “low-intensity” granules (750–2,200 IOD) and the “high-intensity” granules (8,000–10,000 IOD). The data represent compilation of three independent experiments.