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. 2014 Oct 23;289(50):34743–34767. doi: 10.1074/jbc.M114.576702

FIGURE 9.

FIGURE 9.

NaBu increases Sp1/3 transcriptional activity. A, Sp3 and Sp4 expression was unaffected by NaBu treatment. NIE115 cells were incubated with or without 1 mm NaBu for 24 h. Whole cell lysates were prepared and immunoblotted for Sp3, Sp4, or β-actin (loading control). Both short Sp3 (sSp3) and long Sp3 (lSp3) isoforms are shown. B, NaBu treatment did not lead to increased Sp3 DNA binding. NIE115 cells were treated with or without 1 mm NaBu for 24 h, and nuclear extracts from harvested cells were incubated with biotinylated PKCδ promoter probe spanning the GC(1) and GC(2) sites. The presence of Sp3 was detected by immunoblotting analysis. A representative immunoblot is shown. C, stimulation by NaBu of the Sp1/3 transactivational potential. The reporter plasmid pG5-luc, which contains five Gal4-binding sites upstream of a minimal TATA box, and the effector plasmids for Gal4 (pM), Gal4-Sp3 (pM-Sp3), Gal4-Sp3DBD (pM-Sp3DBD), Gal4-Sp1 (pM-Sp1), Gal4-Sp1DBD (pM-Sp1DBD) were cotransfected into NIE115 (left panel) and MN9D (right panel) cells and incubated with or without 1 mm NaBu for 24 h. Luciferase activities were then determined and normalized by cellular protein. Values are expressed as the fold induction of luciferase activity following transfection of the pG5-luc alone without NaBu treatment and are represented as means ± S.E. of three independent experiments performed in triplicate. Above each blue bar is the fold change in activity in the presence of NaBu over that observed in the absence of NaBu. D, effects of overexpression of HDAC isoforms on the NaBu-induced Gal4-Sp1 (left panel) and Gal4-Sp3 (right panel) transactivation. NIE115 cells were cotransfected with reporter plasmid pG5-luc and 8 μg of Gal4-Sp1 or Gal4-Sp3 in combination with 4 μg of HDAC1, HDAC4, HDAC5, or HDAC7 expression plasmids or empty vector control (pcDNA3.1). The cells were then treated with or without NaBu (1 mm) for 24 h, and luciferase activities were determined. Values are expressed as fold induction over the activity obtained following transfection of the Gal4 without NaBu treatment and are represented as means ± S.E. of three independent experiments performed in triplicate. ***, p < 0.001; pCDNA3.1-transfected versus HDACs-transfected samples.