Figure 4: DDX3 up-regulates the HNF4-mediated transactivation of MTP promoter. | Scientific Reports

Figure 4: DDX3 up-regulates the HNF4-mediated transactivation of MTP promoter.

From: RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

Figure 4

(a) Schematic representation of pGL2/MTP-Luc (−611/+87) reporter plasmid. (b) DDX3 potentiates transactivation activity of HNF4 on MTP promoter. MTP promoter (−611~+87) -driven reporter plasmid (0.25 μg) was transfected alone or together with HA-HNF4 and HA-DDX3 expressing constructs in HuH7 and HepG2 by lipofectamine 2000. The total amount of transfected plasmids was adjusted to 2.5 μg/35 mm dish by supplementing with control vector, pcDNA/HA. After 48 hr, cells were collected and subjected to luciferase activity assay. (c) Schematic representation of pMTP(mutant)-Luc reporter plasmid, with the mutated sequences specified (underlined). (d) Destruction of HNF4-responsive elements blocks transactivation activity of HNF4 on MTP promoter. pGL2/MTP-Luc (−611/+87) reporter plasmid (Wild-type; 0.25 μg) or pMTP(mutant)-Luc plasmid (mutant; 0.25 μg) was transfected alone or together with HA-HNF4 expressing construct (0.25 μg) as indicated in HuH7 by lipofectamine 2000. The total amount of transfected plasmids was adjusted to 2.5 μg/35 mm dish by supplementing with control vector, pcDNA/HA. Forty-eight hours later, cells were collected and subjected to luciferase activity assay. (e) Destruction of HNF4-responsive elements blocks synergistic transactivation activity of HNF4/DDX3 on MTP promoter. pMTP(mutant)-Luc reporter plasmid (0.25 μg) was transfected alone or together with HA-HNF4 and HA-DDX3 expressing constructs as indicated amount in HuH7 and HepG2 by lipofectamine 2000. The total amount of transfected plasmids was adjusted to 2.5 μg/35 mm dish by supplementing with control vector, pcDNA/HA. Cells were collected and subjected to luciferase activity assay 48 hr post-transfection. The relative luciferase activity of pGL2/MTP-Luc (−611/+87) reporter alone is arbitrarily taken as one. All data are represented as the average (mean ± S.D.) of at least three independent experiments. (f) ATPase activity of DDX3 is required for its transactivation activity of HNF4 on MTP promoter. HuH7 and HepG2 cells were transfected with pGL2/MTP-Luc (−611/+87) reporter plasmid (0.25 μg) alone or together with indicated amount of plasmids expressing HA-HNF4, HA-DDX3(WT), HA-DDX3(DQAD) and HA-DDX3(AAA). Luciferase activity was measured 48 hr post-transfection.

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