Figure 4

BACH1 affects the expression of MGMT through impairing the association between p53 and SP1.

(A) Western blot analysis of p53 expression in DBTRG-05MG cells transfected with BACH1 expression plasmid or shBACH1-2. (B) Western blot analysis of p53 expression in P-GBM2 cells transfected with BACH1 expression plasmid or shBACH1-2. (C–E) Lysates from GBM cells were incubated in the presence of anti-IgG, or anti-p53, or anti-SP1, or anti-BACH1 antibody. Immunoprecipitated material was subjected to SDS-PAGE, and Westernblot analysis was performed with anti-p53, anti-SP1 and anti-BACH1 antibodies. (F,G) Co-IP was performed using lysates prepared from DBTRG-05MG cells transfected with BACH1, or vector control, or shBACH1-2, or shCtrl using anti-IgG or anti-p53 antibody. And Westernblot analysis was performed with indicated antibodies. (H) DBTRG-05MG cells were separately transfected with wild-type p53, shBAHC1, or BACH1. ChIP assays were performed using anti-IgG or anti-SP1 antibody. The eluted DNA was subjected to qRT-PCR with the specific primer set for the MGMT promoter region. (I) DBTRG-05MG cells stably expressing shp53 were separately transfected with shBAHC1 or BACH1. ChIP assays were performed using anti-IgG or anti-SP1 antibody. The eluted DNA was subjected to qRT-PCR with the specific primer set for the MGMT promoter region. Student’s t-tests and One-way ANOVA were performed. Data are presented as mean ± SEM (**P < 0.01, ^#P > 0.05).