Figure 5.

Pit-1 binding regulates the timing of hPRL transcription. Single-cell transcriptional activity of hPRL WT cells following siRNA Pit-1 knockdown or siRNA negative control, 72 h after transfection. Representative images and single-cell transcriptional traces from siRNA negative control treated hPRL WT (A and C) and hPRL WT, Pit-1 siRNA knockdown cells (B and D). Colored lines represent data from single cells with the thick black line representing the mean response of the population; n = 30 cells (c), n = 22 cells (D). Luciferase activity from each cell was normalized to the average luminescence intensity of all cells in each imaging experiment at time zero. Luciferase activity (E) and Pit-1 protein expression (E inset) of hPRL WT cells following treatment with lipofectamine transfection reagent, siRNA negative control or Pit-1 siRNA knockdown (KD). Estimated transcriptional “on” time determined by SSM, compared between hPRL WT scrambled siRNA, and hPRL WT siRNA Pit-1 knockdown. Bars show SD of the cycle length from 77 and 62 cells, respectively, in 3 experiments per cell type. The duration of an “on phase” is significantly reduced in cells with Pit-1 knockdown (Kolmogorov-Smirnov, *P < 0.05) (F).