For DHPG treated chambers, 500 μM DHPG (0342; Tocris Bioscience) solution was perfused for 10 min at 32-37° C. For AIDA controls, 1 mM AIDA (0904; Tocris Bioscience) was first perfused for 5 min, then 1 mM AIDA with 500 μM DHPG was perfused for 10 min. For Actinomycin D controls, 10 μM of Actinomycin D was incubated within the chambers for 30-45 min, followed by DHPG perfusion as described above. The control treated chambers consisted of vehicle (500 μM NaOH). Following DHPG perfusions, all the chambers were incubated for 20 min at 37° C. Chambers were then fixed and processed for FISH (see below).
