518437.fig.001
Figure 1: RGS5 inducible expression in HeyA8-MDR cells reduces proliferation. (a) HeyA8-MDR pTet dual RGS5 inducible cells were seeded in a multiple 6-well and cultured in medium with tetracycline-free fetal bovine serum without doxycycline for 24, 48, and 72 hours. Cells were harvested; the RNA was isolated and processed for qRT-PCR using primers to detect RGS5 expression resulting from the Tet-Off Advanced inducible system. Without the presence of doxycycline or other members of the tetracycline antibiotics, RGS5 expression was observed. (b) HeyA8-MDR cells were plated in a 96-well plate at a density of 3,000 cells per well. Half of the plate was grown in medium containing regular FBS with doxycycline and the other half containing doxycycline-free media. Cells were then incubated at 37°C for 72 hours to allow for RGS5-inducible gene expression. Prior to fixation, HeyA8 MDR cells were pulse-treated for 1 hour with BrdU and then treated for 4 hours with the indicated cell cycle arrest compounds. Cells were then fixed and stained for proliferation and nuclear morphology. Representative images taken using the Cellomics ArrayScan VTI HCS Reader and are shown here. (c) High-content scanning analysis software was used to determine the average number of cells per field.