Fig. 1.
Fig. 1. RNA interference in hematopoietic cells. / (A) Bcr-abl fusion sequence and schematic representation of b3a2_1 and b3a2_3 siRNAs shifted by only one nucleotide. TT indicates the deoxythymidine dimer as 3' overhang. The arrow marks the fusion between bcr (left) and abl (right) sequences of the b3a2–bcr-abl variant. (B) siRNA mediated the reduction of bcr-abl mRNA expression in TonB cells in the presence or absence of IL-3. Normalized bcr-abl/GAPDH mRNA levels were measured 24 hours after electroporation and are shown compared with control cells treated with GL2/invGL2 control siRNAs (100%). The data represent mean ± SD from 3 independent experiments. (C) Immunoblot of TonB cells treated with doxycycline and siRNAs. TonB cells were induced to express BCR-ABL (lanes 1, 3-6) or not (lane 2) by the addition of doxycycline at 1 μg/mL. Cells were electroporated with b3a2_1 siRNA (lanes 4, 6) or control GL2_siRNA (lanes 3, 5) and were lysed 24 (lanes 3-4) and 60 hours (lanes 5-6) after electroporation, respectively. The upper panel shows an immunoblot with anti-BCR–specific antibodies, and the lower panel shows the same membrane reprobed with anti-Stat5 antibodies as loading control. (D-E) Effects of siRNAs on BCR-ABL–mediated (D) and IL-3–mediated (E) cell proliferation. TonB cells were either electroporated with control GL2_ (red squares) or b3a2_1 (blue-filled circles) siRNA or were left untreated in cultures containing 1 μM STI571 (orange open circles). Viable cells were counted by trypan blue exclusion during suspension cultures after the addition of doxycycline at 1 μg/mL without (D) or with (E) murine IL-3. Cell numbers of b3a2_1- and STI571-treated cells were nearly identical in the presence or absence of IL-3. (F) Inhibition of laminA/C protein expression by siRNAs in normal CD34+ cells. Normal CD34+cells were electroporated with control GL2_ (i,iii) or anti–laminA/C siRNAs (ii,iv). Panels i-ii show immunostaining of laminA/C, and panels iii-iv show nuclear chromatin staining with DAPI (4′6-diamidino-2-phenylindole-2HCl) stain.

RNA interference in hematopoietic cells.

(A) Bcr-abl fusion sequence and schematic representation of b3a2_1 and b3a2_3 siRNAs shifted by only one nucleotide. TT indicates the deoxythymidine dimer as 3' overhang. The arrow marks the fusion between bcr (left) and abl (right) sequences of the b3a2–bcr-abl variant. (B) siRNA mediated the reduction of bcr-abl mRNA expression in TonB cells in the presence or absence of IL-3. Normalized bcr-abl/GAPDH mRNA levels were measured 24 hours after electroporation and are shown compared with control cells treated with GL2/invGL2 control siRNAs (100%). The data represent mean ± SD from 3 independent experiments. (C) Immunoblot of TonB cells treated with doxycycline and siRNAs. TonB cells were induced to express BCR-ABL (lanes 1, 3-6) or not (lane 2) by the addition of doxycycline at 1 μg/mL. Cells were electroporated with b3a2_1 siRNA (lanes 4, 6) or control GL2_siRNA (lanes 3, 5) and were lysed 24 (lanes 3-4) and 60 hours (lanes 5-6) after electroporation, respectively. The upper panel shows an immunoblot with anti-BCR–specific antibodies, and the lower panel shows the same membrane reprobed with anti-Stat5 antibodies as loading control. (D-E) Effects of siRNAs on BCR-ABL–mediated (D) and IL-3–mediated (E) cell proliferation. TonB cells were either electroporated with control GL2_ (red squares) or b3a2_1 (blue-filled circles) siRNA or were left untreated in cultures containing 1 μM STI571 (orange open circles). Viable cells were counted by trypan blue exclusion during suspension cultures after the addition of doxycycline at 1 μg/mL without (D) or with (E) murine IL-3. Cell numbers of b3a2_1- and STI571-treated cells were nearly identical in the presence or absence of IL-3. (F) Inhibition of laminA/C protein expression by siRNAs in normal CD34+ cells. Normal CD34+cells were electroporated with control GL2_ (i,iii) or anti–laminA/C siRNAs (ii,iv). Panels i-ii show immunostaining of laminA/C, and panels iii-iv show nuclear chromatin staining with DAPI (4′6-diamidino-2-phenylindole-2HCl) stain.

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