Figure 1. Knockdown or inhibition of Akt activity upregulates PRB transcriptional activity.

A) PRB-Ishikawa cells were transfected with either siCtrl or siAkt1-3. Following transfection, cells were serum-starved and then treated with either Ethanol Vehicle or 10 nM R5020 for 24 hrs. RNA was extracted and microarray analysis was performed. Heatmap analysis was performed to display the differentially expressed genes. B) Venn Diagram showing the distribution and overlap of differentially expressed genes in the siAkt Vehicle, siCtrl R5020, and siAkt R5020 datasets. C) Gene ontology analysis using GeneGO was performed to determine the key processes enriched in the siAkt R5020 differentially expressed dataset. D) PRB-Ishikawa cells were transfected with either siCtrl or siAkt1-3. Following transfection, cells were serum-starved and then treated with either Vehicle, or 10 nM R5020 for 24 hrs. RNA was extracted and real-time PCR analysis was performed. E) Whole cell protein lysates were also extracted and phospho Akt (S473), pan Akt, and Actin were detected by western blotting. F) PRB-Ishikawa cells were serum-starved overnight and then treated with either DMSO and Ethanol Vehicle, 1 μM MK-2206, 10 nM R5020, or 1 μM MK-2206 + 10 nM R5020 for 24 hrs. RNA was extracted and real-time PCR analysis was performed. G) Whole cell lysates were also extracted and phospho Akt (S473), pan Akt, and Actin were detected by western blotting. H) PRB-Ishikawa cells were transfected with either siCtrl or siPR. Following transfection, cells were serum-starved and then treated with either Vehicle, 1 μM MK-2206, 10 nM R5020, 1 μM MK-2206 + 10 nM R5020 for 24 hrs. RNA was extracted and real-time PCR was performed. I) Whole cell lysates were also extracted and PR and Tubulin were detected by western blotting. Error bars represent SEM of three independent experiments, and *p < 0.05.