Regulation of PP6 expression in Caco-2 cells. (A) Caco-2 cells were seeded at < 30% and > 90% confluency as low and high density cultures. After 4 days levels of PP6 mRNA were assayed by quantitative RT-PCR and the average and standard deviation were plotted (n=3). There was a statistically significant increase in PP6 mRNA relative to GAPDH, used as endogenous control. Quantitative PCR was used to assay the levels of miR31 (B) and miR373 (C) in low and high density Caco-2 cells. Experiments were replicated (n=3) and the mean and standard deviation plotted and analyzed for statistical significance. (D) The ratio of firefly to Renilla luciferase activities in Caco-2 cells co-transfected with a PP6c promoter-reporter and a control plasmid. The ratio was normalized to low density cells in three independent experiments done in triplicate, showing a highly significant increase in PP6 promoter activity in high density cells. (E) Immunoblotting for endogenous PP6c, cyclin D1 and PP2Ac in high density Caco-2 cells treated for the indicated periods of time with 100 μg/ml cycloheximide.