Figure 3

P53 knockdown abolished the effects of BACH1 on TMZ resistance in vitro and in vivo.

(A) GBM cells were transfected with the reporter MGMT-Luc construct or with other plasmids as indicated. β-galactosidase constructs was included in each transfection to normalize transfection efficiency. A luciferase reporter assay was performed to measure MGMT promoter activity. TSS, transcription start site. (B) Schematic illustration of the promoter of the human MGMT gene and the region containing the primers for ChIP assay (left). ChIP assays were performed using anti-IgG or anti-BACH1 antibody. The eluted DNA was subjected to qRT-PCR with the specific primer set for the MGMT promoter region (right). (C) DBTRG-05MG cells transfected with BACH1 or vector was subjected to ChIP assays using the indicated antibodies. The eluted DNA was subjected to qRT-PCR with the specific primer set for the MGMT promoter region. (D) DBTRG-05MG cells transfected with FLAG-p53-wt or FLAG-p53-mut (R273H) was subjected to Co-IP analysis using the indicated antibodies. (E) DBTRG-05MG cells separately transfected with p53-wt, p53-mut (R273H), or empty vector was subjected to ChIP assays 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. (F) Western blot analysis of p53 in GBM cells transfected with p53 shRNA or control shRNA. (G,H) GBM cells cotransfected with BACH1 or shBACH1-2, and shp53 were treated with TMZ(200 μM) for 48 hours. Flowcytometry and colony formation assays were used to measured cells apoptosis and proliferation. (I,J) GBM cells cotransfected with BACH1 or shBACH1-2, and shp53 were treated with TMZ(200 μM) for 48 hours. Western blot analysis of the indicated proteins expression. (K) Representative pseudocolour bioluminescence images of orthotopic tumors bearing vector control or ectopic expression BACH1 P-GBM2 cells stably expressing shp53 after treatment with TMZ on the days as indicated. IHC analysis of MGMT and cleaved caspase-3 expression in intracranial xenografts. (L) survival curve of vector control or ectopic expression BACH1 P-GBM2 cells stably expressing shp53-derived intracranial xenografts treated with TMZ. Student’s t-tests and One-way ANOVA were performed. Data are presented as mean ± SEM (**P < 0.01), scale bar = 100 μm.