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. 2019 Mar 25;10(3):247. doi: 10.3390/genes10030247

Figure 4.

Figure 4

E2F binding motifs function as negative and positive regulatory elements. (A) Promoter-luciferase constructs of GOLPH3 and its derivatives. Mutant motifs are depicted with a white cross. (B) Asynchronous U2OS cells were transfected with the indicated luciferase reporter constructs along with pRL-TK and various amounts of a plasmid expressing E2F1 (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. (C) U2OS cells were transfected with the indicated reporter constructs along with pRL-TK and synchronized in mitosis. Subsequently, cells were released into the cell cycle and luciferase values were calculated as relative values of firefly luciferase activities relative to Renilla activities in each sample. (D) Asynchronous U2OS cells were transfected with the indicated reporter constructs and various amounts of a plasmid expressing E2F1 (50 ng, 250 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 two independent experiments. *, p < 0.05.