Figure 5.

YY1 is involved in regulation of EGFR-mediated GBP1 expression. (A) Schematic representation of cis intact (WT) and mutant (mt) YY1 binding motif in the GBP1 proximal promoter. (B) U87-EGFR cells were transfected with the GBP1 wild-type promoter pGL3-237 and the internal control pRL-TK with or without 200-fold excess of YY1 wild-type or mutant decoy or the YY1 deactivated GBP1 promoter pGL3-237-yy1mt and pRL-TK for 24 h and then serum starved for 24 h before 20 ng/ml EGF treatment for 6 h. Firefly and Renilla luciferase activities were measured, and promoter activity was presented as the fold induction of RLU (values of firefly luciferase unit/values of Renilla luciferase unit) as compared with the control. This result is expressed as the mean of three independent experiments ± SD. #, P < 0.05; *, P < 0.01. (C) U87-EGFR cells were transfected with YY1 or Luc-specific siRNA for 24 h and then transfected with pGL3-237/pRL-TK for 24 h. The cells were serum starved for an additional 24 h before 20 ng/ml EGF or PBS treatment for 6 h. Promoter activity was presented as the fold induction of RLU as compared with the control. The result is presented as mean ± SD of three independent experiments. #, P < 0.05; *, P < 0.01. (D) EMSA analysis. Double-strand YY1 DNA probe was labeled with γ-[³²P]ATP and bound to the nuclear extracts of EGF- and/or SB203580-treated U87-EGFR cells with or without preincubation with a 100-fold excess of YY1 probe or YY1-specific antibody. (E) ChIP analysis of YY1 element from untreated and EGF-treated (100 ng/ml, 30 min) U87-EGFR cells using an antibody specific for YY1 or rabbit IgG control. Input chromatin is presented. PCR was performed to amplify the proximal GBP1 promoter (237 bp). This experiment was repeated twice, yielding identical results. (F) U87-EGFR cells were transfected with YY1 or control siRNA for 24 h and then serum starved for 24 h before 20 ng/ml EGF stimulation for 24 h. GBP1 and YY1 expression were analyzed by Western blot. Data are representative of at least two independent experiments. (G) After 24 h of serum starvation, U87-EGFR cells were treated with DMSO vehicle or 20 µM of p38 inhibitor SB203580 for 1 h followed by 100 ng/ml EGF treatment for 30 min before cell fractionation and Western blot analysis. Data are representative of two independent experiments.