Fig. 2.
Fig. 2. siRNA-mediated inhibition of AML1/MTG8 expression in t(8;21)-positive cells. / (A) Autoradiographs of RNase protection assays. 16 hours after electroporation in the presence of 200 nM siRNAs, cellular RNAs were isolated and analyzed by RNase protection assays. Arrows mark the protected fragments corresponding to AML1/MTG8 with 240 nucleotides in length and to AML1 with 100 nucleotides in length. The electroporated siRNAs are indicated at the top. The relative ratios between AML1/MTG8 and AML1 band intensities, as quantified by phosphoimaging, are shown at the bottom. Lanes 1-3, 4-6, 7-9, and 10-13 represent different experiments not performed at the same time. (B) Real-time RT-PCR analysis of AML1/MTG8 expression in siRNA-treated SKNO-1 and Kasumi-1 cells. Total RNA was isolated 16 hours after electroporation with 200 nM siRNA and analyzed by real-time RT-PCR as described in “Materials and methods.” AML1/MTG8 mRNA levels normalized by GAPDH mRNA levels are shown. Black bars (▪), SKNO-1 cells; hatched bars (▧), Kasumi-1 cells. For siRNA nomenclature and target sites, see Table 1. (C) Dose-dependent suppression of AML1/MTG8 by siAM. Kasumi-1 cells were electroporated with the indicated concentrations of siRNA. Cellular RNAs were isolated 16 hours after electroporation and analyzed by RNase protection assays. Black bars (▪), AML1/MTG8-specific siRNA siAM; hatched bar (▧), control siRNA siGL2. (D) Time course of siAM-mediated AML1/MTG8 suppression. Cellular RNAs were isolated at the indicated days after electroporation with 200 nM siRNA and analyzed by RNase protection assays. Black bars (▪), siAM; hatched bars (▧), siGL2. (E) Effects of siRNAs on AML1/MTG8 and AML1 protein levels. Nuclear extracts were prepared either 3 days (lanes 1-5) or 4 days (lanes 6-11) after electroporation with 200 nM siRNA and analyzed by immunoblotting. The electroporated siRNAs are indicated on top. Arrows on the right mark AML1/MTG8 and AML1 protein bands. Markers are shown on the left. Lanes 1-5, 6-8, and 9-11 represent different experiments not performed at the same time. For siRNA nomenclature and target sites, see Table 1.

siRNA-mediated inhibition of AML1/MTG8 expression in t(8;21)-positive cells.

(A) Autoradiographs of RNase protection assays. 16 hours after electroporation in the presence of 200 nM siRNAs, cellular RNAs were isolated and analyzed by RNase protection assays. Arrows mark the protected fragments corresponding to AML1/MTG8 with 240 nucleotides in length and to AML1 with 100 nucleotides in length. The electroporated siRNAs are indicated at the top. The relative ratios between AML1/MTG8 and AML1 band intensities, as quantified by phosphoimaging, are shown at the bottom. Lanes 1-3, 4-6, 7-9, and 10-13 represent different experiments not performed at the same time. (B) Real-time RT-PCR analysis of AML1/MTG8 expression in siRNA-treated SKNO-1 and Kasumi-1 cells. Total RNA was isolated 16 hours after electroporation with 200 nM siRNA and analyzed by real-time RT-PCR as described in “Materials and methods.” AML1/MTG8 mRNA levels normalized by GAPDH mRNA levels are shown. Black bars (▪), SKNO-1 cells; hatched bars (▧), Kasumi-1 cells. For siRNA nomenclature and target sites, see Table 1. (C) Dose-dependent suppression of AML1/MTG8 by siAM. Kasumi-1 cells were electroporated with the indicated concentrations of siRNA. Cellular RNAs were isolated 16 hours after electroporation and analyzed by RNase protection assays. Black bars (▪), AML1/MTG8-specific siRNA siAM; hatched bar (▧), control siRNA siGL2. (D) Time course of siAM-mediated AML1/MTG8 suppression. Cellular RNAs were isolated at the indicated days after electroporation with 200 nM siRNA and analyzed by RNase protection assays. Black bars (▪), siAM; hatched bars (▧), siGL2. (E) Effects of siRNAs on AML1/MTG8 and AML1 protein levels. Nuclear extracts were prepared either 3 days (lanes 1-5) or 4 days (lanes 6-11) after electroporation with 200 nM siRNA and analyzed by immunoblotting. The electroporated siRNAs are indicated on top. Arrows on the right mark AML1/MTG8 and AML1 protein bands. Markers are shown on the left. Lanes 1-5, 6-8, and 9-11 represent different experiments not performed at the same time. For siRNA nomenclature and target sites, see Table 1.

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