Fig. 3.

Sp1 down-regulation mediated ISO inhibition of CD44 transcription. a Total RNA isolated from T24T cells treated with 20 μM ISO for indicated time periods was subjected to RT-PCR to determine cd44 mRNA expression. b Both CD44 luciferase reporter and pRL-TK were co-transfected into T24T cells, and the transfectants were treated with 20 μM ISO for indicated times. Luciferase activity was evaluated as described in “Materials and methods”. *Significant difference (p < 0.05). c Indicated transcription factors in T24T cells were determined by Western Blot after cells were treated with 20 μM ISO for indicated times. d GFP-Sp1 plasmid or vector was transfected into T24T cells; the transfectants were extracted and subjected to Western blot to determine the protein expressions indicated. e, f Sp1 was responsible for ISO inhibition of CD44 transcription. g T24T cells were stably transfected with CD44 promoter-driven luciferase or the Sp1-binding site mutant, and the stable transfectants were treated with 20 μM ISO. Luciferase activity was evaluated as described in “Materials and methods”. h ChIP assay was carried out with anti-GFP antibody. DNA was extracted from GFP agarose or control IgG. Total sonicated nuclei were used as Input control. Specific Sp1 binding DNA regions of the CD44 promoter were then amplified by PCR. PCR products were separated over 2% agarose gels that were then stained with ethidium bromide and examined under UV light. i T24T(vector) and T24T(GFP-Sp1) cells were treated with/without ISO (20 μM) to determine sphere formation ability. *Significant difference (p < 0.05). j T24T(nonsense) and T24T(shSp1) cells were used in sphere formation assay as described in “Materials and methods”. *Significant difference (p < 0.05)