FIGURE 4.

Both Sp1 and NF-κB are required for the transcription of miR-365. A, shown is a schematic representation of the binding sites for Sp1 and NF-κB. The mutated sequences are indicated in lowercase. B, HEK293 cells were transfected with the mutated miR-365 promoter reporters. At 24 h post-transfection, cells were stimulated with poly(dAT:dAT) as described in Fig. 3B. Luciferase activity was measured at 24 h after stimulation. Results are represented as -fold induction over non-stimulated control and are expressed as the mean ± S.D. for triplicate determinations where each experiment is representative of three separate experiments. **, p < 0.01, as compared with the control group. C, TSS-1097-Luc reporter was co-transfected into HEK293 cells with Sp1 specific siRNA or scrambled siRNA. Luciferase activities were measured 24 h post-transfection. D, TSS-1097-Luc reporter was transfected into HEK293 cells. At 24 h post-transfection, cells were treated with BAY11-7082 (15 μm) for 1 h, and then poly(dAT:dAT) (2 μg/ml) was transfected into HEK293 cells in some wells. E, the Sp1 or p65 expression constructs were co-transfected with TSS-1097-Luc reporter or individual mutant into HEK293 cells, respectively. Luciferase activities were measured at 24 h post-transfection. F, TSS-1097-Luc reporter (0.1 μg) was co-transfected with a total of 0.6 μg of a DNA mixture composed of pEGFP-N1-Sp1 and/or pEGFP-C1-p65 and/or empty vector at the indicated dose. Luciferase activities were measured 24 h post-co-transfection. In all cases results were represented as -fold induction over control. Data were represented as the mean ± S.D. (n = 3). **, p < 0.01.