Fig. 7.

Sp1 activates the transcription of SOD2 in U87MG TMZ-resistant cells. (A) Schematic of the SOD2 core promoter showing several Sp1- and AP-2-binding sites. (B) U87MG cells were treated with 100 µM TMZ for one month. After treatment, eleven random single cell-derived clones from these surviving cells were analyzed by Western blotting with the indicated antibodies. (C) Control and TMZ-resistant GBM xenografts were analyzed by IHC using anti-Sp1 or anti-SOD2 antibodies. The dotted lines in the top panel outline areas with high Sp1 and SOD2 expression. The bottom panel is a magnified view of the inset red box from the top panel. (D) SOD2 mRNA levels in U87MG (Con) cells and resistant cells (TMZ-R) were quantified by real-time PCR. (E) After treatment with different doses of MA, SOD2 expression in TMZ-resistant cells was analyzed by Western blotting. (F) The level of Sp1 binding to the SOD2 promoter in TMZ-resistant clones (#9 and #10) was assessed using a DNA affinity precipitation assay (DAPA), with a biotin-labeled DAPA probe that contains two Sp1-binding sites (P: SOD2) as shown in (A). (*p < 0.05; **p < 0.01; ***p < 0.001).