The −1377 G > A variant affects SP1 binding and transcriptional activity of the CD95 promoter. (A-B) EMSA illustrating binding of nuclear proteins to the polymorphic site in the CD95 core promoter. (A) ³²P-labeled CD95 −1377 DNA probe (G allele, lanes 1, 2, 5, 6, 9 to 11; A allele, lanes 3, 4, 7, and 8) was incubated with 15 μg HL60 or NB4 nuclear extract (lanes 2, 4, 6, 8, and 11). Incubation with anti–human SP1 antibody (lane 10) generated a supershifted complex confirming the identity of the DNA binding protein as SP1. SP1 binding was quantified using densitometry and is shown in panel B. Error bars represent the mean ± SEM from 3 independent experiments, and statistical analysis was performed using the unpaired Student t test for SP1 binding. (C-D) siRNA-mediated knockdown of SP1 gives rise to a concomitant reduction in CD95 protein expression. RNA interference of SP1 in HL60 or NB4 cells was performed using an siRNA specific to SP1 (sc-29487, Santa Cruz Biotechnology). Cells were electroporated with SP1 or MLL-AF4 siRNA (negative control), and the degree and specificity of SP1 knockdown and effect on CD95 protein expression were assessed by immunoblot analysis 8 hours after transfection (C). (D) The results of quantification using densitometry. Results were normalized against β-actin levels. Error bars represent the mean ± SEM from 3 independent experiments, and statistical analysis was performed using the unpaired Student t test. (E) Reporter gene assay illustrating the effect of the −1377 variant on CD95 promoter activity. Promoter activity was analyzed using the Dual-Luciferase Reporter assay system, with reporter vector luminescence (firefly [FF]) normalized against control luminescence (TK-renilla [Rh]). Luciferase activity was assessed from 6 independent clones for each −1377 variant. Error bars represent the mean ± SEM from 6 independent experiments. Statistical analysis was performed using the unpaired Student t test.