Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: The interaction between Ahi-1 and DNM2 depends on SH3-PRD recognition within endosomal compartments. ( a ) Schematic representations of four Ahi-1 and DNM2 constructs, including HA-tagged full-length Ahi-1 (HA-Ahi-1), HA-tagged SH3 domain-deleted Ahi-1 (HA-SH3Δ), Myc-tagged full-length DNM2 (Myc-DNM2) and Myc-tagged proline rich domain-deleted DNM2 (Myc-PRDΔ). 293T cells co-transfected with indicated constructs were stained with anti-HA (green) and anti-Myc (red) antibodies. DAPI was used to stain the nuclei. Representative images are shown. ( b , c ) 293T cells co-transfected with indicated constructs were stained with anti-HA (green) and anti-EEA1 (red, b ) or anti-LAMP-1 (red, c ) antibodies. Images were acquired using a magnification of × 60 by confocal microscopy. The white scale bar represents 5 μm.

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques: Construct, Transfection, Staining, Confocal Microscopy

Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: Increased expression of DNM2 in CD34 + CML stem/progenitor cells and lentiviral-mediated knockdown of DNM2 in BCR–ABL + cells affects the JAK2/STAT5 pathway. ( a ) Quantitative real-time PCR analysis of the transcript levels of DNM2 in CD34 + cells purified from normal bone marrow (NBM), IM responders (IM R) and IM nonresponders (IM NR). DNM2 transcript levels were normalized to the control gene β2M , and bars represent the mean of data for each group. Comparison of the transcript levels of DNM2 in three subpopulations from IM NR ( n =8, red) and IM R ( n =5, blue). ( b ) Western blot analysis of phosphorylation and protein expression levels of DNM2 and other proteins in DNM2 knockdown K562 cells (shDNM2A and shDNM2B) and BV173 cells (shDNM2A). The densitometry values of protein expression changes are indicated as compared to SHC control.

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Purification, Western Blot

Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: Lentiviral-mediated knockdown of DNM2 impairs the survival of CD34 + CML stem/progenitor cells and sensitizes these cells to TKIs. ( a ) Cell proliferation of control (SHC) or DNM2-knockdown CD34 + CML cells with or without 5 μ M IM (left) and MitMAB (right). ( b ) Cell viability (left) and apoptosis (right) assays in SHC or DNM2-knockdown CD34 + CML cells with or without 5 μ M IM or 150 n M DA treatments. Values shown are the mean±s.e.m. * P <0.05, *** P <0.001.

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques:

Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: Lentiviral-mediated knockdown of DNM2 impairs the survival of very primitive CD34 + CML stem/progenitor cells and identification of the AHI-1–BCR–ABL–DNM2 protein complex. ( a ) Numbers and types of colonies produced by transduction of CD34 + IM-nonresponder cells ( n =3) with either a control (SHC) or shDNM2A construct in semisolid culture medium with or without 5 μ M IM or 150 n M DA. ( b ) Long-term culture-initiating cell (LTC-IC) analysis of colony-forming cell (CFC) outputs in the same transduced cells cultured for 6 weeks in the presence of stromal cells with or without 5 μ M IM or 150 n M DA. ( c ) Co-immunoprecipitation assays in K562 and K562 IM-resistant cells (IMR, left) and BCR–ABL-transduced and BCR–ABL/Ahi-1 co-transduced BaF3 cells (middle). Protein extracts were subjected to anti-DNM2 immunoprecipitation and then immunoblotted with anti-c-Abl antibody or anti-DNM2 antibody. 293T cells were co-transfected with HA-Ahi-1, BCR–ABL and Myc-DNM2 (or Myc-DNM2 PRDΔ) or HA-Ahi-1 SH3Δ, BCR–ABL and Myc-DNM2 (or Myc-DNM2 PRDΔ, right). Cells were stained with anti-Myc (red), anti-HA (purple) and anti-c-Abl (green) antibodies. DAPI was used to stain the nuclei. Images were acquired using a magnification of × 60 by confocal microscopy. The white scale bar represents 5 μm.

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques: Produced, Transduction, Construct, Cell Culture, Immunoprecipitation, Transfection, Staining, Confocal Microscopy

Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: Western blot analysis of the AHI-1–BCR–ABL–DNM2 protein complex, with DNM2 phosphorylation by BCR–ABL. ( a , b ) Co-immunoprecipitation assays in various BCR–ABL + cell lines with or without IM for 24 h. Parental BaF3 cells were cultured with mIL-3 (10 ng) but not Ahi-1 or BCR–ABL-transduced BaF3 cells. All protein extracts were immunoprecipitated with anti-DNM2 antibody and immunoblotted with a pan-anti-p-Tyr antibody (4G10) or anti-DNM2 antibody. ( c ) Co-immunoprecipitation assays in BCR–ABL/Myc-DNM2 co-transfected 293T cells cultured with or without 5 μ M IM for 24 h. Protein extracts were immunoprecipitated with anti-Myc antibody and then immunoblotted with a pan-anti-p-Tyr antibody (4G10) or anti-Myc antibody. ( d ) Co-immunoprecipitation assays of UT7 BCR–ABL T315I (UT7 B/A T315I) cells with or without ponatinib (20 n M ) or ABL001 (4 μ M ) treatment for 24 h. All protein extracts were immunoprecipitated with an anti-DNM2 antibody and immunoblotted with a pan-anti-p-Tyr antibody (4G10).

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques: Western Blot, Immunoprecipitation, Cell Culture, Transfection

Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: Suppression of DNM2 or inhibition of BCR–ABL affects transferrin uptake in CD34 + CML cells from IM nonresponders. CD34 + CML cells transduced with either control (SHC) or shDNM2A and cultured for 24 h ( a ) with or without IM or ( b ) CD34 + CML cells from the same patients treated with IM or MitMAB alone or in combination were stained with Alexa Fluor 647-conjugated transferrin and transferrin uptake was determined by confocal microscopy. Intracellular transferrin signals were quantified and normalized to SHC control cells or untreated cells ( n =3 per group, respectively). The white scale bar represents 50 μm. Values shown are the mean±s.e.m. ** P <0.01.

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques: Inhibition, Transduction, Cell Culture, Staining, Confocal Microscopy

Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: Suppression of DNM2 or inhibition of BCR–ABL affects ROS production in primary CD34 + CML cells from IM nonresponders. ( a , b ) ROS production was determined using CellROX deep red reagents in CD34 + CML cells transduced with either a control (SHC) or shDNM2A and cultured ( a ) with or without IM or ( b ) CD34 + CML cells from the same patients treated with IM or MitMAB alone or in combination. Intracellular ROS accumulation was quantified and normalized to the signals detected in SHC or untreated control cells by confocal microscopy ( n =3 per group, respectively). Representative images are shown. The white scale bar represents 50 μm. Values shown are the mean±s.e.m. * P <0.05, ** P <0.01.

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques: Inhibition, Transduction, Cell Culture, Confocal Microscopy

Journal: Leukemia

Article Title: A novel AHI-1–BCR-ABL–DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

doi: 10.1038/leu.2017.108

Figure Lengend Snippet: The effects of suppression of DNM2 on key autophagy regulators in primary CD34 + CML cells and a model of biological functions of the ABD complex in CML cells. ( a ) Western blot analysis of ULK-1, Beclin-1, LC3-II and p62 in CD34 + IM-nonresponder cells ( n =3) with knockdown of DNM2 as indicated. The densitometry values of protein expression changes are indicated. Bar graph represents the quantification of the protein levels of ULK-1, Beclin-1, LC3-II and p62 relative to Actin and SHC controls in DNM2 knockdown CD34 + CML cells. Values shown are the mean±s.e.m. ** P <0.01. ( b ) Model of the mechanism by which the ABD protein complex deregulates three essential cellular activities—endocytosis, ROS production and autophagy—in CML stem/progenitor cells, resulting in increased LSC survival and genomic stability, but reduced TKI response of these cells. Knockdown or pharmaceutical inhibition of DNM2 activities and TKI treatments to destabilize this complex perturbs these key cellular properties.

Article Snippet: The pGFP-C-lenti vector (OriGene), containing the non-targeting sequence or DNM2 shRNA constructs, and the pRRL-PPT-SF-GFP-pre vector were used as templates to amplify the U6 promoter-shRNAs and the SFFV promoter, respectively.

Techniques: Western Blot, Expressing, Inhibition