Figure 3.

GOLPH3 gene is regulated by E2F factors at the transcriptional level. (A) Promoter-luciferase construct of GOLPH3. The nucleotide positions of the promoter construct are numbered relative to the transcription start site. (B) Activation of GOLPH3 promoter-driven firefly luciferase reporter construct during the cell cycle. U2OS cells were transfected with pGP3 wild type construct (pGP3-WT) or pGL2 empty construct along with pRL-TK. Cells were subsequently synchronized in mitosis with Thy-Noc, and released into the cell cycle. Values are represented as firefly luciferase activities relative to Renilla luciferase activities of each sample. (C) Activation of GOLPH3 promoter-driven construct in asynchronous cells. Asynchronously growing U2OS cells were transfected with pGP3-WT construct or pGL2 empty construct along with pRL-TK. Values are represented as firefly luciferase activities relative to Renilla luciferase activities of each sample. (D) Ectopic E2F1-3 expression induces GOLPH3 promoter activity. Asynchronously growing U2OS cells were transfected with pGP3-WT and increasing amounts of E2F1-3 (50 ng, 150 ng, 250 ng) per well in a 12-well plate. Values are represented as luciferase activities relative to pGP3-WT activity of cells transfected with empty pCMV control. (E) Ectopic E2F7 expression represses GOLPH3 promoter activity. Asynchronously growing U2OS cells were transfected with pGP3-WT and increasing amounts of E2F7 (50 ng, 150 ng, 250 ng) in the absence (left panel), or in the presence of E2F1 co-expression (150 ng). Values are represented as luciferase activities relative to pGP3-WT activity of cells transfected with empty pCMV control. Shown are the results of a representative experiment of three independent experiments. *, p < 0.05.