Sp1 is sufficient to induce CYP1B1-mediated effects.

(A) ZEB2 and E-cadherin mRNA were measured by RT-PCR and (B) E-cadherin promoter activity was determined by luciferase assay after Sp1 overexpression in MCF-7 cells. (C) Key proteins in Wnt/β-catenin signaling and (D) EMT-related factors were measured by western blot after Sp1 induction in MCF-7 cells. (E and F) Similar to Fig 5C and 5D, but following Sp1 inhibition. (G and H) Also similar to Fig 5C and 5D, but in MCF-7 cells co-transfected with CYP1B1 overexpression vector and Sp1 siRNA. (I and J) MCF-7 cells treated with DMBA and mithramycin A for 24 h following pre-treatment with mithramycin A for 1 h. (I) Key factors in Wnt/β-catenin signaling and (J) EMT were determined by western blot. (K) PCNA in MCF-7 cells, (L) β-catenin in MCF-10A cells, (N) ZEB2 in MCF-7 cells were observed by confocal microscopy after treatment with 5 μM DMBA and 100 nM mithramycin A for 24 h. (M) β-catenin proteins in nuclear or cytosolic fraction of MCF-10A cells were measured following treatment with 5 μM DMBA and 100 nM mithramycin A for 24 h. Cells were pre-treated with mithramycin A for 1 h prior to DMBA. (O) Relative promoter activity of β-catenin/TCF/LEF, ZEB1, and TWIST1 was determined by dual-luciferase assay after treatment with DMBA and mithramycin A. As before, cells were pre-treated with mithramycin A for 1 h prior to DMBA. Data are representative of experiments in triplicate. (*p≤0.05)