shrna dnm2 against exon potential off-target in mouse and dnm2  (ACGT Inc)

Journal: Cancer research

Article Title: Metastasis Suppressors NME1 and NME2 Promote Dynamin 2 Oligomerization and Regulate Tumor Cell Endocytosis, Motility and Metastasis.

doi: 10.1158/0008-5472.CAN-19-0492

Figure Lengend Snippet: A Two-way co-immunoprecipitation of NME1 and DNM2 was performed on vector and NME1 overexpressing MDA-MB-231T cells using rabbit anti-NME1 and goat anti-DNM2 antibody. B Two-way co-immunoprecipitation in vector and NME1 overexpressing MDA-MB-435 cells was performed with the above antibodies. C& D Two-way co-immunoprecipitation of NME2 and DNM2 was performed on vector and NME2 overexpressing MDA-MB-231T cells using rabbit anti-NME and mouse anti-DNM2 antibodies. E Co-immunoprecipitation of NME1 and DNM2 was performed on a panel of breast cancer cell lines with varying metastatic status (low to high) using anti-NME antibody for immunoprecipitation. F DNM2 construct map showing deletion in PRD domain [Construct 1: Contains full PRD domain, Construct 2: truncated PRD domain (Δ42 amino acid), Construct 3: deleted PRD domain]. All the constructs contain PH and GED domain. G In vitro co-immunoprecipitation of NME1 was performed with DNM2 or with deletion constructs using anti-NME antibody. Data are representative of three independent experiments.

Article Snippet: Gene silencing DNM2 targeting shRNAs were purchased from GeneCopoeia (Stable selection marker: Hygromycin and Reporter gene: mcherry). shRNAs were transfected into MDA-MB-231T- vector and NME1 overexpressing cells and were selected using Hygromycin.

Techniques: Immunoprecipitation, Plasmid Preparation, Construct, In Vitro

Journal: Cancer research

Article Title: Metastasis Suppressors NME1 and NME2 Promote Dynamin 2 Oligomerization and Regulate Tumor Cell Endocytosis, Motility and Metastasis.

doi: 10.1158/0008-5472.CAN-19-0492

Figure Lengend Snippet: A, B Vector and NME overexpressing (NME1 and NME2) MDA-MB-231T cells were plated in chamber slides and treated with a non-cytotoxic dose (2 μM) of dynamin inhibitor (Iminodyn-22) or vehicle for 24 hrs. Cells were then incubated with Transferrin-Alexa-594 (A) or pHrodo™ Red-EGF dye (B) for 15 min., fixed in PFA and nuclei stained with DAPI (blue). C Vector and NME overexpressing MDA-MB-231T cells were plated in chamber slides and cultured with and without 2 μM Iminodyn-22 for 24 hrs. Cells were then fixed in PFA and immunofluorescence was performed with AP2 alpha to detect clathrin coated pits (red). Cells are outlined by dotted lines and nuclei counterstained with DAPI. D-F Effect of DNM2 shRNA. D Western blot of MDA-MB-231T vector and NME1 transfectants, each transfected with a scrambled shRNA (−) or DNM2 shRNA (+). β-actin was used as loading control. E Vector and NME overexpressing MDA-MB-231T cells stably transfected with scrambled or shDNM2 were incubated with Transferrin-Alexa-633 for 15 min. Following acid washes, cells were fixed in PFA and nuclei were visualized by DAPI (blue); staining was visualized using confocal microscopy with representative images shown. F Quantification of Transferrin-Alexa-633 uptake in the transfectants described in (E) using Zeiss software (ZEN). For all images, merged views are shown. Scale bar-10 μm. All experiments shown are representative of 3 replicates and statistical significance was calculated by a 1-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001).

Article Snippet: Gene silencing DNM2 targeting shRNAs were purchased from GeneCopoeia (Stable selection marker: Hygromycin and Reporter gene: mcherry). shRNAs were transfected into MDA-MB-231T- vector and NME1 overexpressing cells and were selected using Hygromycin.

Techniques: Plasmid Preparation, Incubation, Staining, Cell Culture, Immunofluorescence, shRNA, Western Blot, Transfection, Stable Transfection, Confocal Microscopy, Software

Journal: Cancer research

Article Title: Metastasis Suppressors NME1 and NME2 Promote Dynamin 2 Oligomerization and Regulate Tumor Cell Endocytosis, Motility and Metastasis.

doi: 10.1158/0008-5472.CAN-19-0492

Figure Lengend Snippet: A Vector and NME transfected MDA-MB-231T cells stably transfected with scrambled or shDNM2, described in the legend to Figure 3, were assessed for motility in Boyden chambers as described in the legend to Figure 4. B Representative photographs of the Boyden chamber membrane underside showing migrated tumor cells. C Migration in scratch assays of the same cells, as described in the legend to Figure 4. D Representative photographs of the scratch invaded. E 7.5 × 105 MDA-MB-231T cells expressing vector and NME1 overexpression with or without DNM2 knockdown (Vector-scr, Vector-shDNM2, NME1-scr, NME1-shDNM2) were injected into the lateral tail vein of athymic nude mice (each group, n=11). At 9 weeks post-injection, the mice were sacrificed, and the lungs were fixed in Bouin’s solution followed by H&E staining. A representative image of each group is presented with arrows pointing to metastases. F All metastases in the lung sections were counted and presented as scatter plot showing median with interquartile range. Each dot represents a single mouse. All in vitro experiments shown are representative of (motility n=4 and migration n=3) replicates and statistical significance was calculated by a 1-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001). For metastasis 1-way ANOVA (nonparametric) test was performed comparing median across all the groups with P < 0.05 considered significant (*).

Article Snippet: Gene silencing DNM2 targeting shRNAs were purchased from GeneCopoeia (Stable selection marker: Hygromycin and Reporter gene: mcherry). shRNAs were transfected into MDA-MB-231T- vector and NME1 overexpressing cells and were selected using Hygromycin.

Techniques: Plasmid Preparation, Transfection, Stable Transfection, Migration, Expressing, Over Expression, Injection, Staining, In Vitro

Journal: Cancer research

Article Title: Metastasis Suppressors NME1 and NME2 Promote Dynamin 2 Oligomerization and Regulate Tumor Cell Endocytosis, Motility and Metastasis.

doi: 10.1158/0008-5472.CAN-19-0492

Figure Lengend Snippet: A Western blot of DNM2 and NME. Recombinant DNM2 (3.5 μg) was incubated with wild type or mutant recombinant, partially purified NME1s (1.5 μg) in HCB75 buffer for 15 min at 22°C. Each mixture was ultracentrifuged at 214,000 × g for 15 min at 4°C and the resulting pellets (P) and supernatants (S) fractions were processed on western blots. In this assay, DNM2 monomers remained in the supernatant (S) while oligomers precipitated into the pellet (P). B A GTPase assay was performed on DNM2 (3 μg), alone or incubated with wild type NME1, NME1P96S or NME1H118F (1μg) in the presence of liposomes for 15 min, followed by addition of 4 mM GTP and color reagent. OD at 620 nm was measured after 30 min incubation. C Recombinant DNM2 (3.5 μg) was incubated with recombinant NME2 (1.5 μg) as per the oligomerization assay protocol described above and was processed on western blot. D GTPase assay was performed on DNM2 (3 μg), alone or incubated with NME2 (1μg) as per the GTPase protocol described above. All experiments shown are representative of three (n=3) replicates and statistical significance was calculated by a 1-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001).

Article Snippet: Gene silencing DNM2 targeting shRNAs were purchased from GeneCopoeia (Stable selection marker: Hygromycin and Reporter gene: mcherry). shRNAs were transfected into MDA-MB-231T- vector and NME1 overexpressing cells and were selected using Hygromycin.

Techniques: Western Blot, Recombinant, Incubation, Mutagenesis, Purification

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

Journal: The Journal of Cell Biology

Article Title: Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts

doi: 10.1083/jcb.201401137

Figure Lengend Snippet: Dynamin expression increases in early OCPs, and dynamin depletion impairs osteoclast multinucleation. (A) Western blot analysis of BMM cell lysates stimulated with RANKL for the indicated days to evaluate expression levels of dynamin, NFATc1, Src, cathepsin K, CHC, and actin (loading control) during RANKL-induced osteoclast differentiation. (B) Western blot analysis of osteoclasts with the indicated antibodies and time points. Cells from littermate control mice Dnm1 flox/flox ;Dnm2 flox/flox were used as a control for tamoxifen treatment. (C) Representative images of TRAP-stained osteoclasts differentiated from bone marrow of Dnm1 flox/flox ;Dnm2 flox/flox mice and CreER; Dnm1 flox/flox ;Dnm2 flox/flox littermates. Osteoclast multinucleation was quantified by counting the number of TRAP+ cells with one to two nuclei (left) or three or more nuclei (right). The inset is an enlarged view of the boxed region. Data are means ± SD (error bars) from five independent experiments. *, P < 0.001. Bars, 200 µm. (D) Images of TRAP-stained CTL and DKO osteoclasts after culturing with RANKL for 1 d. Note that DKO cells are round and have less prominent filopodia, compared to CTL cells. Bars, 10 µm.

Article Snippet: Dnm2 shRNA lentiviral particles were purchased from Sigma-Aldrich, and CHC shRNA constructs were from OriGene.

Techniques: Expressing, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts

doi: 10.1083/jcb.201401137

Figure Lengend Snippet: Dynamin PRD and PH domains are required for the fusion of OCPs. (A) TRAP staining of transduced Dnm -DKO osteoclasts reexpressing vector, dynamin 1 (Dnm1), dynamin 2 (Dnm2) WT, and Dnm2 mutants including a GTPase mutant K44A, a PH domain mutant K562E, and a splice variant ΔPRD. Bars, 200 µm. (B) Quantification of TRAP-positive cells with more than three nuclei for fusion rescue efficiency. One-way ANOVA (P < 0.001) and a Student’s t test were used to analyze the data. a , significantly different from vector (P < 0.001); b , significantly different from Dnm2 (P < 0.034).

Article Snippet: Dnm2 shRNA lentiviral particles were purchased from Sigma-Aldrich, and CHC shRNA constructs were from OriGene.

Techniques: Staining, Plasmid Preparation, Mutagenesis, Variant Assay

Journal: The Journal of Cell Biology

Article Title: Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts

doi: 10.1083/jcb.201401137

Figure Lengend Snippet: Dynamin depletion also prevents primary myoblast fusion. Primary myoblasts isolated from Dnm1 flox/flox ;Dnm2 flox/flox mice and CreER; Dnm1 flox/flox ;Dnm2 flox/flox littermates were cultured in differentiation medium for 4 d with or without 4OHT. (A) Dynamin depletion in creER;Dnm-Dfl cells after 4OHT treatment is shown by Western blot analysis. (B) DNM-DKO myoblasts showed a defect in myotube formation (phase-contrast microscopy), and the syncytium formation extent was quantified and normalized to Dfl cells. Error bars indicate SEM. *, P < 0.01.

Article Snippet: Dnm2 shRNA lentiviral particles were purchased from Sigma-Aldrich, and CHC shRNA constructs were from OriGene.

Techniques: Isolation, Cell Culture, Western Blot, Microscopy