FIGURE 7. Mutation of Sp1 binding sites abrogates p53 effects on MnSOD transcription.

A, schematic representation of the MnSOD basal promoter and specific mutations of its Sp1 sites in the construct were shown as black boxes. B, either p53 expression-vector (4 µg) or empty vector (equivalent amount) along with MnSOD promoter-driven luciferase reporter vector were transfected in HepG2 cells. Parallel transfection experiments were performed using MnSOD promoter-driven luciferase reporter vectors, where three or five Sp1 binding sites in the promoter region were mutated. After 24 h of co-transfection, cells were washed and grown for another 24 h, and then cells were treated with TPA (100 nm) for 12 h. Cell lysates were collected, and luciferase activity was measured as a determinant of MnSOD gene transcription. DMSO, Me₂SO. C, EMSA was performed to check whether there was interference due to the direct binding of p53 to Sp1 binding sites. No binding of purified p53 protein (EMSA grade) to the Sp1 consensus sequence was detected. Each data point represents the mean ± S.D. of three experiments. **, p < 0.01 compared with the control; #, p < 0.01 compared with corresponding treatment; *, p < 0.05.