Activity and specificity of MLL-AF4 siRNAs. (A) SiRNA scan of the MLL-AF4 mRNA fusion site. MLL-AF4 mRNA levels normalized by GAPDH mRNA levels are shown. Target sites of the indicated siRNAs were moved by one single nucleotide from the AF4 part to the MLL part of the fusion site. Total RNA was isolated 24 hours after electroporation with 500 nM of siRNA and analyzed by real time RT-PCR. One of 2 experiments yielding similar results is shown. (B) Time course of MLL-AF4 depletion. Total RNA was isolated at the indicated time points after electroporation with 750 nM siRNA. Real time RT-PCR was performed as in panel A. □ indicates Mock; ▪, siMA6; ▧, siMM. (C) Depletion of MLL-AF4 protein upon siRNA transfection. Total cell lysates were isolated 48 hours after electroporation with 500 nM siRNA. MLL-AF4 was detected with an antibody targeting the C-terminus of AF4. GAPDH served as a loading control and for normalization. Normalized MLL-AF4 protein levels are indicated at the bottom. (D) Effects of the MLL-AF4 siRNA siMA6 and a mismatch control siMM on MLL-AF4, AF4, and MLL mRNA levels in SEM cells. Analysis was performed as in panel A. □ indicates MLL; ▪, AF4; ▧, MLL-AF4. (E) Effects of the MLL-AF4 siRNAs siMA6 and siMARS and a mismatch control siMM on MLL-AF4, AF4, and MLL mRNA levels in RS4;11 cells. Analysis was performed as in panel A. siMA6 is homologous to the MLL-AF4 variant expressed in SEM cells; siMARS targets the variant present in RS4;11 cells. □ indicates MLL; ▪, AF4; ▧, MLL-AF4. (F) MLL-AF4 siRNAs do not induce an interferon response. SEM cells were transfected with the indicated RNAs. PolyIC (7.5 μg/mL) served as a positive control for the induction of the interferon response genes OAS1 and STAT1. Analysis was performed as in panel A.