Figure 3.

BNIP3 binds to a specific region in the DR5 promoter. (a) Oligonucleotide probes containing four different putative BNIP3 DNA binding sequences (Supplementary Table 1) from the promoter region of DR5 (R1-4), beads alone (beads) and a non-specific promoter region were incubated with recombinant BNIP3 protein. Input indicates lysate alone without beads or DNA. Interestingly, only the oligonucleotide containing region 2 of DR5 showed binding with BNIP3. (b) This pull-down experiment was repeated using lysates from HEK293 cells that stably express BNIP3 under the control of a Tet-ON promoter. Without tetracycline, BNIP3 expression is not seen in the beads alone, R1, R2 or the non-specific control. However, in the presence of tetracycline, binding is again observed in region 2. (c) A 60-bp fragment of the DR5 promoter was cloned into the luciferase reporter gene construct and then transfected into U251 parental cells, and U251 stably expressing shRNA against BNIP3. All cells were co-transfected with a beta-galactosidase vector for control of transfection efficiency. Luciferase activity was measured by a Softmax Pro Luminometer. These results are representative of three independent experiments. (d) HEK293 cells were transfected with BNIP3-NLS expression vector or vector alone (pcDNA3) in combination with luciferase vector containing the DR5 promoter region 1 or 2. Cells were lysed and western blotted for BNIP3 and actin as a loading control. (e) Transfected HEK293 cells were also measured for luciferase activity as described above. These results are representative of the three independent experiments. * represented statistical significance from three independent experiments