Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effects of Delta1 overexpression on Notch activation and keratinocytes differentiation. (A) Schematic representation of the Delta constructs. (B) Anti DeltaD immunofluorescence staining of human keratinocytes transduced with DeltaFl. Scale bar: 20μm. (C) Hes1 luciferase reporter activity (upper panel) in J2-3T3 cells transduced with empty vector (ev), or Delta constructs. Fold induction relative to Renilla control is shown. Mean of 4 experiments ± SEM. (lower panels) Western blot of lysates of J2-3T3 cells used in the luciferase assay, probed with antibodies to DeltaD and Actin (loading control). (D) Clonal growth of keratinocytes transduced with empty vector or Delta constructs. (E) Clonal growth of wild type keratinocytes on a feeder layer of J2-3T3 cells transduced with empty vector (ev) or Delta constructs. (F-H) Epidermis reconstituted on DED by keratinocytes transduced with empty vector (ev,F,I), DeltaFl (G,J) or DeltaDS (H,K). Sections were stained with H&E (F-H) or antibody to Keratin 10 (green) with DAPI nuclear counterstain (blue) (I-K). Scale bar: 50μm.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Over Expression, Activation Assay, Construct, Immunofluorescence, Staining, Transduction, Luciferase, Activity Assay, Plasmid Preparation, Control, Western Blot

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effect of Delta1 expression on clonal growth of keratinocytes Keratinocytes were transduced with the retroviral vectors indicated and cultured on a wild type feeder layer. WT: cells transduced with empty vector. Data are mean ± s.d. of triplicate dishes from at least 3 experiments. CFE: colony forming efficiency.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Expressing, Transduction, Retroviral, Cell Culture, Plasmid Preparation, Clone Assay

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effect of Delta1 expressing feeders on clonal growth of wild type keratinocytes Wild type keratinocytes (K-WT) were cultured on feeder layer s of J2 3T3 cells transduced with the retroviral vectors shown. Data are mean ± s.d. of triplicate dishes in at least 3 experiments. CFE: colony forming efficiency.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Expressing, Cell Culture, Transduction, Retroviral, Clone Assay

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: The Delta1 PDZ domain binds syntenin and promotes keratinocyte cohesiveness. (A) GFP labelled clones formed by keratinocytes expressing the Delta constructs shown 5 days after seeding within a confluent sheet of unlabelled WT keratinocytes. Scale bar: 20 μm. (B) Percentage of cohesive colonies. Data are mean of 5 experiments ± SD. Values significantly different from WT control are marked with asterisks (student’s t-test: P<0.001*** or P<0.05**). (C) Western blots of A431 cells transfected with empty vector (ev) or the Delta constructs shown following pull down with GST (control; right hand panel) or GST-syntenin (left hand panel). Blots were probed with anti-ZfDelta or anti-GST. (D) Western blot of untransfected A431 cell lysates following pull down with GST or GST-syntenin (GST-syn). Blot was probed with antibody to human Delta1. Total cell lysates (Lys) served as positive control. (E) A431 cell lysates immunoprecipitated with antibodies against syntenin (α-syn) or laminin5 (α-Lam) followed by western blotting with anti Delta 1 antibody. Lys: lysate subjected to western blotting without prior immunoprecipitation. (F) Immunofluorescence staining of human primary keratinocytes with anti syntenin antibody (green). DAPI was used as a nuclear counterstain (blue). Scale bar: 10 μm. (G) Immunofluorescence staining of human interfollicular epidermis with an anti syntenin antibody. Horizontal lines mark position of putative stem cell clusters. Scale bar: 50 μm.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Clone Assay, Expressing, Construct, Control, Western Blot, Transfection, Plasmid Preparation, Positive Control, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Functional consequences of syntenin knockdown. (A) Western blot of keratinocytes transduced with empty vector (ev) or syntenin RNAi (Rnai1) probed with anti syntenin (α-syn) or, as a loading control, anti tubulin (α-tub). (B) Hes1 luciferase reporter activity in J2-3T3 cells transduced with empty vector (ev) or the Delta constructs shown, in the presence or absence of syntenin RNAi. Fold induction relative to Renilla control is shown. Mean of 3 experiments ± SEM. Values significantly different from ev control or Fl are marked with asterisks (student’s t-test: P<0.001*** or P<0.05**). (C) Clonal growth of keratinocytes transduced with DeltaFl or Delta DS and control (Ctrl) or syntenin Rnai1. (D-G) GFP labelled clones formed by human keratinocytes expressing DeltaFl alone (Fl) (D) or in combination with syntenin Rnai1(E), or formed by mouse keratinocytes with endogenous Delta1 levels (Dll1flox/flox)(F) or lacking Delta1 (K5Cre × Dll1flox/flox)(G). Clones were photographed 5 days after seeding within a confluent sheet of unlabelled WT keratinocytes (human or mouse). Scale bar: 100 μm. (H) Percentage of cohesive colonies formed by GFP labelled keratinocytes. Data are mean of 3 experiments ± SD (student’s t-test: P < 0.001***).

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Functional Assay, Knockdown, Western Blot, Transduction, Plasmid Preparation, Control, Luciferase, Activity Assay, Construct, Clone Assay, Expressing

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effects of syntenin knock down or Delta1 mutation on cell surface expression of Delta mutants. (A-F) Keratinocytes transduced with DeltaFl and pRetrosuper control vector (A-C) or syntenin Rnai (D-F) were double labelled with anti DeltaD (green A,D) and anti syntenin (red B,E). Right hand panels are merged images of middle and left hand panels (C,F). Scale bars: 20μm. (G-O) Primary human keratinocytes transduced with DeltaFl, DeltaDS or DeltaVA were incubated with an anti-Delta antibody at 4°C (t = 0), transferred to fresh medium at 37°C for the times indicated. Scale bar: 50μm. (P) Quantitation of % cells with Delta antibody at the plasma membrane. Data are means of 3 independent experiment ±SEM. 100 cells of each type were counted per experiment. (Q) Western blot of keratinocytes transduced with ev, DeltaFl, DeltaDS and DeltaVA constructs, probed with antibodies to Zf-Delta, syntenin and actin.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Knockdown, Mutagenesis, Expressing, Transduction, Control, Plasmid Preparation, Incubation, Quantitation Assay, Clinical Proteomics, Membrane, Western Blot, Construct

Journal: eLife

Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

doi: 10.7554/eLife.32860

Figure Lengend Snippet: ( A, B ) HEK293T cells were transfected with Citrine-MPDZ together with HA-tagged DLL1, HA-tagged DLL1 ΔPDZ (lacking the PDZ-binding site), Flag-tagged DLL4 or Flag-tagged DLL4 ΔPDZ . Antibodies against Citrine were used to immunoprecipitate Citrine-MPDZ. HA and FLAG-tagged proteins as well as MPDZ were detected by immunoblot (IB). Scheme shows structures of the constructs used for co-immunoprecipitation. Input, 10% of the immunoprecipitate. Cit-MPDZ, Citrine-MPDZ; IP, immunoprecipitation; neg.ctrl., negative control. ( C, D ) MPDZ was co-expressed with either DLL1 or DLL4 in primary endothelial cells (HUVEC). DLL1 and DLL4 were pulled down by using specific antibodies. DLL1, DLL4 and MPDZ were detected by immunoblot (IB). Input, 5% of the immunoprecipitate. IP, immunoprecipitation; neg.ctrl., negative control. ( E ) HUVEC were either transduced with adenovirus expressing GFP (ctrl) or MPDZ (MPDZ). Expression level of Notch target genes HEY1 , HEY2 and HES1 were analyzed by qPCR 48 hr after transduction. Data are presented as mean ±SD. n = 4; *, p<0.05; **, p<0.01 unpaired Student’s t-test. ( F ) Scheme of the co-culture Notch reporter assay. IMCD3 cells expressing the Notch ligand DLL4 were co-cultured with CHO-N1-CIT cells carrying a Notch luciferase reporter construct. The IMCD3 sender cells were modified by expression of MPDZ or an empty vector control. After 48 hours, cells were lysed and the light emission of the luciferin and the Renilla luciferase activities were measured. Signaling activity is calculated by normalizing the luciferase signal with the Renilla signal. Data are presented as mean ± SEM. n = 5; *, p<0.05 unpaired Student’s t-test. 10.7554/eLife.32860.005 Figure 1—source data 1. Source data of qantitative PCR analysis related to .

Article Snippet: Cells were suspended (10 6 cells/ml) and incubated with different fluorophores coupled to primary antibodies against DLL1 (FAB1818A, R & D Systems), DLL4 (FAB1506A, R & D Systems), Dll4 (563802, BD Biosciences), CD34 (553733, BD Biosciences) for 20 min on ice.

Techniques: Transfection, Binding Assay, Western Blot, Construct, Immunoprecipitation, Negative Control, Transduction, Expressing, Co-Culture Assay, Reporter Assay, Cell Culture, Luciferase, Modification, Plasmid Preparation, Control, Activity Assay

Journal: eLife

Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

doi: 10.7554/eLife.32860

Figure Lengend Snippet: ( A, B ) Dll1 and Dll4 were immunoprecipitated by using specific antibodies from lysates of mouse kidneys. Mpdz was detected by immunoblot (IB). Input, 10% of the immunoprecipitate. IP, immunoprecipitation; neg.ctrl., negative control. ( C, D ) HEK293T control cells (293T sh-ctrl) as well as MPDZ-silenced HEK293T cells (293T sh-MPDZ) were transfected with SYNJ2BP and HA-tagged DLL1 or Flag-tagged DLL4. For immunoprecipitation either a DLL1 or a Flag-tag antibody was used and DLL1, DLL4 and SYNJ2BP were detected by Western Blotting. Input, 10% of immunoprecipitate; IB, Immunoblot; IP, Immunoprecipitation; neg.ctrl., negative control.

Article Snippet: Cells were suspended (10 6 cells/ml) and incubated with different fluorophores coupled to primary antibodies against DLL1 (FAB1818A, R & D Systems), DLL4 (FAB1506A, R & D Systems), Dll4 (563802, BD Biosciences), CD34 (553733, BD Biosciences) for 20 min on ice.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Control, Transfection, FLAG-tag

Journal: eLife

Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

doi: 10.7554/eLife.32860

Figure Lengend Snippet: ( A ) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression level of Notch target genes HEY1 , HEY2 and HES1 were analyzed by qPCR 48 hr after transduction. Data are presented as mean ±SD. n ≥ 3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student’s t-test. ( B ) Cardiac endothelial cells were isolated from Mpdz fl/fl and Mpdz ΔEC mice by magnetic beads bound with CD31 antibodies. Expression levels of Notch target genes Hey1 and Hey2 were analyzed by qPCR. Data are presented as mean ±SD. n = 3; *, p<0.05; ***, p<0.001 unpaired Student’s t-test. ( C ) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression levels of DLL1 and DLL4 were analyzed by immunoblotting 48 hr after transduction. β-actin served as loading control. Data are presented as mean ±SD. n ≥ 3; n.s., not significant. ( D ) HUVECs were either transduced with adenovirus expressing GFP (ctrl) or with adenovirus expressing MPDZ. Expression levels of DLL1 and DLL4 were analyzed by immunoblotting 48 hr after transduction. β-actin served as loading control. Data are presented as mean ±SD. n ≥ 3; n.s., not significant. ( E ) Lung endothelial cells were isolated from Mpdz fl/fl and Mpdz ΔEC mice by CD31 magnetic beads. Protein amounts of Dll1 and Dll4 were analyzed by immunoblotting. β-actin served as loading control. Data are presented as mean ±SD. n = 3; n.s., not significant. 10.7554/eLife.32860.007 Figure 2—source data 1. Source data of qantitative PCR analysis related to .

Article Snippet: Cells were suspended (10 6 cells/ml) and incubated with different fluorophores coupled to primary antibodies against DLL1 (FAB1818A, R & D Systems), DLL4 (FAB1506A, R & D Systems), Dll4 (563802, BD Biosciences), CD34 (553733, BD Biosciences) for 20 min on ice.

Techniques: Transduction, Expressing, shRNA, Isolation, Magnetic Beads, Western Blot, Control

Journal: eLife

Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

doi: 10.7554/eLife.32860

Figure Lengend Snippet: ( A ) DLL1 and DLL4 full length and such lacking the PDZ-binding site (ΔPDZ) constructs containing a mCherry tag were expressed in HUVEC. Cells were stained with antibodies against DLL1 or DLL4. DLL1 and DLL4 surface expression of mCherry positive cells was analyzed by flow cytometry. n = 3; *, p<0.05; **, p<0.01 unpaired Student’s t-test. ( B ) Endothelial cells were isolated from Mpdz +/+ embryos and Mpdz -/- littermates at embryonic day E11.5. Cells were purified by CD31 magnetic dynabeads and stained with anti-CD34 and anti-Dll4 antibodies for flow cytometric analysis. n = 4; *, p<0.05; unpaired Student’s t-test. ( C, D ) HEK293T control cells (293T sh-ctrl) as well as MPDZ-silenced HEK293T cells (293T sh-MPDZ) were transfected with Nectin-2 and HA-tagged DLL1 or Flag-tagged DLL4. For immunoprecipitation a Nectin-2 antibody was used and HA-tagged DLL1, Flag-tagged DLL4 and MPDZ were detected by western Blotting. Input, 10% of immunoprecipitate. IB, Immunoblot; IP, Immunoprecipitation; neg.ctrl., negative control. 10.7554/eLife.32860.010 Figure 3—source data 1. Source data of FACS analysis related to .

Article Snippet: Cells were suspended (10 6 cells/ml) and incubated with different fluorophores coupled to primary antibodies against DLL1 (FAB1818A, R & D Systems), DLL4 (FAB1506A, R & D Systems), Dll4 (563802, BD Biosciences), CD34 (553733, BD Biosciences) for 20 min on ice.

Techniques: Binding Assay, Construct, Staining, Expressing, Flow Cytometry, Isolation, Purification, Control, Transfection, Immunoprecipitation, Western Blot, Negative Control

Journal: eLife

Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

doi: 10.7554/eLife.32860

Figure Lengend Snippet: ( A, B ) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Cells were cultured under sparse conditions ( A ) or confluent conditions ( B ). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2 and counterstained with DAPI. Images were acquired with the confocal microscope LSM 700. Arrow indicates co-localization of DLL1/4 with Nectin-2 at the cell membrane. Arrow head indicates diminished co-localization at the cell membrane. Scale bar: 10 µm. ( C ) HUVECs were either transfected with control siRNA (si-ctrl) or with siRNA against Nectin-2 (si-Nectin-2). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2. Images were acquired with the confocal microscope LSM 700. Arrow indicates localization of DLL1/4 at the cell membrane.Scale bar: 10 µm.

Article Snippet: Cells were suspended (10 6 cells/ml) and incubated with different fluorophores coupled to primary antibodies against DLL1 (FAB1818A, R & D Systems), DLL4 (FAB1506A, R & D Systems), Dll4 (563802, BD Biosciences), CD34 (553733, BD Biosciences) for 20 min on ice.

Techniques: Transduction, Expressing, shRNA, Cell Culture, Staining, Microscopy, Membrane, Transfection, Control

Journal: eLife

Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

doi: 10.7554/eLife.32860

Figure Lengend Snippet:

Article Snippet: Cells were suspended (10 6 cells/ml) and incubated with different fluorophores coupled to primary antibodies against DLL1 (FAB1818A, R & D Systems), DLL4 (FAB1506A, R & D Systems), Dll4 (563802, BD Biosciences), CD34 (553733, BD Biosciences) for 20 min on ice.

Techniques: shRNA, Western Blot, Recombinant, Cloning, Plasmid Preparation, Mutagenesis, Clone Assay