Fig. 4. Single-cell analyses of the transcriptional response of the PRL array to ligands. (A,B) Cells were (A) mock-transfected or (B) transiently transfected with GFP-ER expression plasmid. Cells in B were treated with vehicle or the indicated ligands (10 nM) for 2.5 hours, processed for FISH and imaged as described in Materials and Methods. GFP-ER signal is shown in green, mRNA FISH signal is shown in red. Overlay includes DAPI-stained nuclei (blue). (C) The FISH signals at the array were quantified as described in Materials and Methods and shown as bar graphs. Treatments (30 minutes, white bars and 2 hours, black bars) are indicated below the graphs; `Non' and `Transfected' indicate mock- and transfected cells, respectively. FISH signals at the array were quantified in 20 cells for each treatment as relative intensity of the cells treated with vehicle and are shown as bar graphs. While there is constitutive level of FISH signal (A), it increases significantly in cells with arrays demarcated with GFP-ER. ^*P=0.007, E2-treated GFP-ER-expressing cells at 30 minutes compared with vehicle controls. ^**P<0.001, E2-treated GRP-ER-expressing cells at 2 hours compared with vehicle controls. ^***P=0.0016, non-transfected cells compared with GFP-ER-expressing cells. ^#P=0.0378, 30 minutes compared with cells treated 2 hours with E2. P<0.001, antagonist-treated GFP-ER-expressing cells versus vehicle controls.