Figure 4

Sp1 attenuates DNMT3a-mediated DNA methylation and increases CYP17A1 expression in TMZ-resistant cells. (a) After treatment with 5-aza-cytidine for 24 h, the cells were collected and subjected to western blotting and MSP. (b) In the presence or absence of Sp1 knockdown, the cells were treated with 5-aza-cytidine for 24 h and subjected to western blotting. The quantitative result for CYP17A1 (**P<0.01) is shown in the right panel. (c) After treatment, the protein lysates were prepared and subjected to DAPA using the S1 probe as described in ‘Materials and Methods’. Probe-binding beads were analyzed by western blotting. (d) Effect of DNMT3a knockdown on CYP17A1 expression. The quantitative results for CYP17A1 (***P<0.001) are shown in the right panel. (e) Effects of GFP-DNMT3a overexpression on CYP17A1 expression in the absence or presence of GFP-Sp1 overexpression. Quantitative results for CYP17A1 (*P<0.05, **P<0.01) are shown in the lower panel. (f) After GFP-Sp1 overexpression, the binding activities of Sp1 and DNMT3a on the CYP17A1 promoter were analyzed with DAPA. (g) DNA-binding activities of DNMT3a in U87MG and U87MG-R were analyzed with DAPA. DAPA, DNA affinity precipitation assay; MSP, methylation-specific PCR.