Figure 11.
TSA induced TSPO promoter activity through box GC3, and Sp1 and Sp3 differentially alter TSA effect on TSPO promoter in MDA-MB-231 and MCF-7 cells.(A) A series of luciferase reporter plasmids with mutations targeting putative binding motifs GC.1, GC.2, GC.3, GC.4, and GC.5 was constructed in the context of the −121/+66 promoter construct, along with the full length promoter and a −76 mutation that does not target a known element, were transiently expressed in MDA-MB-231 and MCF-7 cells treated (black bars) or not (white bars) with TSA for 24 h. Luciferase activity was measured 24 h after TSA treatment. Empty vector was used as a control. Results are derived from three independent experiments (n = 9) **p < 0.01, ***p < 0.001 vs. the wild-type promoter. (B) Co-transfection of MDA-MB-231 cells with increasing amounts of pPacSp1 or pPacSp3 along with F11 construct and treating on not with TSA for 24 h before measuring luciferase activity. (C) Co-transfection of MCF-7 cells with increasing amounts of pPacSp1 or pPacSp3 along with F11 construct and treating on not with TSA for 24 h before measuring luciferase activity. Sp1, Sp3, and Sp4 reamain bound to the endogenous TSPO promoter in intact (D) MDA-MB-231 and (E) MCF-7 cells after 24 h treatment with TSA. DNA from MDA-MB-231 and MCF-7 cells treated with TSA was precipitated with antibodies specific for Sp1, Sp3, and Sp4 in ChIP assays. DNA was amplified by QRT-PCR using primers specific for the proximal area of the TSPO promoter. Normal rabbit IgG served as a negative control. Data were analyzed as percent of input and fold enrichment compared to IgG, and are derived from three independent experiments (n = 9). **p < 0.01, ***p < 0.001 vs. control IgG.