Figure 4

Rapamycin treatment significantly reduces the binding of YY1 to the αSMA promoter element. (A) EMSA analysis of a DNA probe corresponding to the putative YY1 binding site in the αSMA promoter. Labeled probes were incubated with nuclear extracts isolated from untreated and treated AML cells with different concentrations of rapamycin (0–40 nM). (B) Treatment of AML cells with 20-40 nM of rapamycin significantly reduced YY1 in the nuclear extracts compared to untreated cells and cells. Mutant of YY1 oligonucleotide in TTT to AAA base substitutions showed no binding to YY1 promoter-specific DNA complexes (Lane 5 was moved from its original position on the gel). (C) The specificity of binding of the DNA/protein complex to YY1 was demonstrated by adding YY1 antibody to the reaction mixture. Including the YY1 antibody in the reaction results in markedly reduced the specific DNA/protein complex. (D) A reporter plasmid of αSMA promoter driving expression of the luciferase contained wild type or mutant of YY1 was transfected into AML cells. Control Renilla reporter gene was co-transfected into the cells using LipofectAMINE Plus Reagent^TM. Luciferase activity was determined using the Luciferase Reporter Assay System by a luminometer and normalized by Renilla reporter activity. Histograms represent means ± SE (n = 6). Significant difference from cells transfected with wild type YY1 is indicated by *P < 0.01.