Figure 1
Figure 1. Identification novel NOTCH2 and FLT3 splice variants. (A) This figure displays NOTCH2 and FLT3 RT-PCR agarose gel electrophoresis results. (B) This figure displays AML-associated splicing patterns of NOTCH2 and FLT3 splice variants. After cloning and sequencing experiments, novel splice variant sequences were identified through alignment with published sequences for human NOTCH2 and FLT3 mRNA (gi:317008612 and gi:178535 NCBI, respectively). NOTCH2-Va and NOTCH2-Vb are the result of complete deletions of exon 12 (111 bp) or exons 17 (120 bp) and 18 (153 bp), respectively. These splicing aberrations did not cause any frame shifts. FLT3-Va is a result of skipping exon 7 (140 bp) and a 76-bp deletion of exon 8 at the 5′ end, whereas FLT3-Vb was due to skipping exons 5 (130 bp) and 7 (140 bp), with partial deletions of exons 6 (33-bp deletion at the 3′ end of exon 6) and 8 (48-bp deletion at the 5′ end of exon 8). FLT3-Vc appears to be most severely affected by splicing events compared with other FLT3 splice variants. FLT3-Vc is a result of skipping of exons 5 (130 bp), 6 (128 bp), and 7 (140 bp) and a 26-bp deletion of exon 8 at the 5′ end. As a result of these aberrations 240 bp are spliced out from FLT3-Va, 351 bp from FLT3-Vb, and 398 bp from FLT3-Vc. Thus, these aberrations did not cause a frame shift on FLT3-Va and FLT3-Vb transcripts, whereas FLT3-Vc transcripts were subjected to a frame shift. For both NOTCH2 and FLT3, the splice variant transcripts retained their original start codons and conserved signal sequences. Also, bioinformatics and alignment analysis showed that exons affected by aberrant splicing events mapped to extracellular domains of the Notch2 and Flt3 protein sequences. As a result of the splicing alterations, 3 EGF-like domains were entirely deleted from the Notch2 protein and 2 were partially affected. Splicing alterations caused deletion of the entire Ig-like C2 type domain on the Flt3 protein. In the figure, we zoomed out gene segments of NOTCH2 and FLT3 that we cloned and sequenced. In the figure, yellow-boxed exons are those that are affected by the splicing events. (C) HEK293T cells were transiently transfected with NOTCH2-FL-GFP, NOTCH2-Va-GFP, NOTCH2-Vb-GFP, FLT3-FL-GFP, FLT3-Va-GFP, FLT3-Vb-GFP, or GFP backbone plasmids. Seventy-two hours after transfection cells were stained using the live cell nuclear staining reagent (Life Technologies) that includes DAPI. Cells were visualized under the Zeiss 710 confocal laser-scanning microscope. On the figure, GFP signal shown in green, and DAPI signal in blue. HEK293T cells transfected with NOTCH2 and FLT3 splice-variants were stained with anti-Notch2-PE (16F11, eBioscience) and anti-Flt3-APC (BV10A4H2, eBioscience) antibodies, then membrane expression of these variants was evaluated by flow cytometry. On the flow cytometry histograms, gray peaks represent HEK293T cells untransfected and unstained, pink peaks represent HEK293T cells untransfected and stained with anti-Notch2-PE or anti-Flt3-APC antibodies, red peaks represent HEK293T cells transfected with NOTCH2-FL, FLT3-FL, or splice variants tagged with GFP. These cells were stained with anti-Notch2-PE or anti-Flt3-APC antibodies and staining was determined in GFP gated cells. IgG stained controls are provided in Supplemental Figure 1A.

Identification novel NOTCH2 and FLT3 splice variants. (A) This figure displays NOTCH2 and FLT3 RT-PCR agarose gel electrophoresis results. (B) This figure displays AML-associated splicing patterns of NOTCH2 and FLT3 splice variants. After cloning and sequencing experiments, novel splice variant sequences were identified through alignment with published sequences for human NOTCH2 and FLT3 mRNA (gi:317008612 and gi:178535 NCBI, respectively). NOTCH2-Va and NOTCH2-Vb are the result of complete deletions of exon 12 (111 bp) or exons 17 (120 bp) and 18 (153 bp), respectively. These splicing aberrations did not cause any frame shifts. FLT3-Va is a result of skipping exon 7 (140 bp) and a 76-bp deletion of exon 8 at the 5′ end, whereas FLT3-Vb was due to skipping exons 5 (130 bp) and 7 (140 bp), with partial deletions of exons 6 (33-bp deletion at the 3′ end of exon 6) and 8 (48-bp deletion at the 5′ end of exon 8). FLT3-Vc appears to be most severely affected by splicing events compared with other FLT3 splice variants. FLT3-Vc is a result of skipping of exons 5 (130 bp), 6 (128 bp), and 7 (140 bp) and a 26-bp deletion of exon 8 at the 5′ end. As a result of these aberrations 240 bp are spliced out from FLT3-Va, 351 bp from FLT3-Vb, and 398 bp from FLT3-Vc. Thus, these aberrations did not cause a frame shift on FLT3-Va and FLT3-Vb transcripts, whereas FLT3-Vc transcripts were subjected to a frame shift. For both NOTCH2 and FLT3, the splice variant transcripts retained their original start codons and conserved signal sequences. Also, bioinformatics and alignment analysis showed that exons affected by aberrant splicing events mapped to extracellular domains of the Notch2 and Flt3 protein sequences. As a result of the splicing alterations, 3 EGF-like domains were entirely deleted from the Notch2 protein and 2 were partially affected. Splicing alterations caused deletion of the entire Ig-like C2 type domain on the Flt3 protein. In the figure, we zoomed out gene segments of NOTCH2 and FLT3 that we cloned and sequenced. In the figure, yellow-boxed exons are those that are affected by the splicing events. (C) HEK293T cells were transiently transfected with NOTCH2-FL-GFP, NOTCH2-Va-GFP, NOTCH2-Vb-GFP, FLT3-FL-GFP, FLT3-Va-GFP, FLT3-Vb-GFP, or GFP backbone plasmids. Seventy-two hours after transfection cells were stained using the live cell nuclear staining reagent (Life Technologies) that includes DAPI. Cells were visualized under the Zeiss 710 confocal laser-scanning microscope. On the figure, GFP signal shown in green, and DAPI signal in blue. HEK293T cells transfected with NOTCH2 and FLT3 splice-variants were stained with anti-Notch2-PE (16F11, eBioscience) and anti-Flt3-APC (BV10A4H2, eBioscience) antibodies, then membrane expression of these variants was evaluated by flow cytometry. On the flow cytometry histograms, gray peaks represent HEK293T cells untransfected and unstained, pink peaks represent HEK293T cells untransfected and stained with anti-Notch2-PE or anti-Flt3-APC antibodies, red peaks represent HEK293T cells transfected with NOTCH2-FL, FLT3-FL, or splice variants tagged with GFP. These cells were stained with anti-Notch2-PE or anti-Flt3-APC antibodies and staining was determined in GFP gated cells. IgG stained controls are provided in Supplemental Figure 1A.

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