Fig. 3

Fstl1 mediates epigenetic regulation of MGMT through histone deacetylation. a–c MGMT expression was blocked using a lentivirus-mediated RNA interference (shMGMT) or MGMT inhibitor O⁶-benzylguanine (O⁶-BG, 20 μM) in D54 cells stably expressing Fstl1 plasmid or vector control. The cells were subsequently treated with TMZ (200 μM) for 48 h. Flow cytometry (a) and colony formation assays (c) were used to measure cells apoptosis and proliferation, western blot analysis of the indicated proteins expression (b). d 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. e 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-Fstl1 antibody. The eluted DNA was subjected to qRT-PCR with the specific primer set for the MGMT promoter regions (right). f D54 cells transfected with Fstl1 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. g, h Western blot and qRT-PCR analysis of the indicated genes expression in D54 cells transfected with Fstl1 or vector control. i D54 cells were separately transfected with Fstl1 or vector control. ChIP assays were performed using anti-IgG, or specific antibodies against H3K9Ac. The eluted DNA was subjected to qRT-PCR with the specific primer set for the MGMT promoter region. Student’s t tests were performed. Data are presented as mean ± SEM (**P < 0.01, ^#P > 0.05)