Journal: eLife

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

doi: 10.7554/elife.32860

Figure Lengend Snippet: Figure 1. MPDZ interacts with DLL1 and DLL4. (A, B) HEK293T cells were transfected with Citrine-MPDZ together with HA-tagged DLL1, HA-tagged DLL1DPDZ (lacking the PDZ-binding site), Flag-tagged DLL4 or Flag-tagged DLL4DPDZ. 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) Figure 1 continued on next page

Article Snippet: Cells were suspended (106 cells/ml) and incubated with different fluoro- phores 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

Journal: eLife

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

doi: 10.7554/elife.32860

Figure Lengend Snippet: Figure 3. MPDZ recruits DLL1 and DLL4 to Nectin-2. (A) DLL1 and DLL4 full length and such lacking the PDZ-binding site (DPDZ) 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+/+

Article Snippet: Cells were suspended (106 cells/ml) and incubated with different fluoro- phores 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

Journal: Cell Discovery

Article Title: Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells

doi: 10.1038/s41421-022-00467-2

Figure Lengend Snippet: a Organoid aggregate images at specified time points. b Immuno-phenotypes of hemogenic endothelial cells (iHECs, defined as CD73 – DLL4 + CD31 + CD34 + CD144 + ) on Day 9. c Immuno-phenotypes of hematopoietic progenitor cells (iHPCs, defined as CD235a – CD45 + CD34 + CD43 + ) on Day 16. d Immuno-phenotypes of iNK cells (CD45 + CD3 – CD56 + CD16 +/– ) on Day 27. e The images of NK cells separated from organoid aggregates on Day 27. f The dynamic quantities of iNK cells (CD45 + CD3 – CD56 + ) at the indicated time points ( n = 3 each group). g Statistics of yields of iNK cells (Day 27) induced from different hPSC lines. n = 4 each group. Data were collected from four batches of induction experiments using five human ESC lines (hPSC-1, hPSC-2, hPSC-3, hPSC-4, and hPSC-5) and one iPSC line (hPSC-6). One point represents the mean of iNK cell output from three repeats per batch induction experiment. h The residual OP9 feeder cells at the selected time points ( n = 3 each group). Two-tailed independent t -test. Mann-Whitney U test ( f ), and one-way ANOVA ( g ).

Article Snippet: The following antibodies were used: BRACHYURY (R&D System, IC2085A), APLNR (R&D System, FAB8561A-025), CD3 (Biolegend, HIT3a), CD16 (Biolegend, 3G8), CD31 (Biolegend, WM59), CD34 (Biolegend, 581), CD43 (BD Biosciences, 1G10), CD45 (Biolegend, HI30), CD56 (Biolegend, HCD56), CD73 (eBioscience, AD2), CD144 (Biolegend, BV9), CD235a (Biolegend, HI264), DLL4 (Miltenyi, REA1065), NKp30 (Biolegend, P30-15), NKp44 (Biolegend, P44-8), NKG2D (Biolegend, 1D11), CD319 (Biolegend, 162.1), NKG2A (Biolegend, S19004C), CD96 (Biolegend, NK92.39), CD94 (BD Biosciences, HP-3D9), CD69 (Biolegend, FN50), TRAIL (Biolegend, RIK-2), FasL (Biolegend, NOK-1), GzmB (Biolegend, QA18A28), Perforin (Biolegend, dG9).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Scientific Reports

Article Title: HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed by thalidomide

doi: 10.1038/srep27280

Figure Lengend Snippet: ( A ) The expression of HIF-1α and HIF-2α in gastrointestinal vascular malformations and normal vessels. Red arrow: strongly positive; Black arrow: weakly positive; Blue arrow: negative. ( B ) Percentages of positive and negative vessels in GIVM and normal tissues. ** P < 0.01.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA), which was blocked using 5% milk for 2 h, and incubated with rabbit-anti-HIF-2α, VEGF, NOTCH1, DLL4, Ang2, and β-actin antibodies (all rabbit polyclonal antibodies from KangChen Bio-tech, Shanghai, China) at 4 °C overnight.

Techniques: Expressing

Journal: Scientific Reports

Article Title: HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed by thalidomide

doi: 10.1038/srep27280

Figure Lengend Snippet: ( A ) Western blot determinations of HIF-1α and HIF-2α expression at different time points of hypoxia. * P < 0.05, ** P < 0.01 vs. 0 hour. ( B ) The effect of HIF-1α and HIF-2α overexpression on the expression of VEGF, Notch1, DLL4, and Ang2. Western blot and RT-PCR demonstrated that HIF-1α and HIF-2α overexpression increased the expression of VEGF, Notch1, DLL4, and Ang2 protein and mRNA. * P < 0.05, ** P < 0.01 vs. control. ( C ) Influence of HIF-1α and HIF-2α overexpression on angiogenesis 6 and 24 h after transfection of Lenti-HIF-1α and Lenti-HIF2α. Tube formation was enhanced 6 and 24 h after transfection. ** P < 0.01 vs. control. ( D ) Fluorescence microscope observations of subintestinal vein sprouting in normal and HIF-2α-overexpressing zebrafish. *Indicates subintestinal vascular sprouts. HIF-2α overexpression significantly increased the number of subintestinal vascular sprouts. ** P < 0.01 vs. control plasmid. ( E ) Dual luciferase reporter gene assay demonstrated that HIF-2α enhanced VEGF promoter activity. ** P < 0.01 vs. control plasmid.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA), which was blocked using 5% milk for 2 h, and incubated with rabbit-anti-HIF-2α, VEGF, NOTCH1, DLL4, Ang2, and β-actin antibodies (all rabbit polyclonal antibodies from KangChen Bio-tech, Shanghai, China) at 4 °C overnight.

Techniques: Western Blot, Expressing, Over Expression, Reverse Transcription Polymerase Chain Reaction, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Luciferase, Reporter Gene Assay, Activity Assay

Journal: Scientific Reports

Article Title: HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed by thalidomide

doi: 10.1038/srep27280

Figure Lengend Snippet: ( A ) Immunofluorescence indicated that HIF-1α and HIF-2α expression was down-regulated by thalidomide at different concentrations. ( B ) Western blots demonstrated that the expression of HIF-1α and HIF-2α decreased with 100 and 200 μg/ml of thalidomide. * P < 0.05, ** P < 0.01. ( C ) Western blots demonstrated that thalidomide at 200 μg/ml inhibited the expression of HIF-1α and HIF-2α in HUVECs after hypoxic treatment for 24, 36, and 48 h. * P < 0.05, ** P < 0.01. ( D ) Fluorescence microscope observations of the effect of thalidomide at different concentrations on vascular development in zebrafish with HIF-2α overexpression. ** P < 0.01 vs. HIF-2α.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA), which was blocked using 5% milk for 2 h, and incubated with rabbit-anti-HIF-2α, VEGF, NOTCH1, DLL4, Ang2, and β-actin antibodies (all rabbit polyclonal antibodies from KangChen Bio-tech, Shanghai, China) at 4 °C overnight.

Techniques: Immunofluorescence, Expressing, Western Blot, Fluorescence, Microscopy, Over Expression

Journal: Cancer treatment reviews

Article Title: Targeting the Wnt/beta-catenin Pathway in Cancer: Update on Effectors and Inhibitors

doi: 10.1016/j.ctrv.2017.11.002

Figure Lengend Snippet: Examples of drugs/agents that inhibit Wnt/β-Catenin Signaling

Article Snippet: MEDI0639 , DLL4-specific Notch antibody , MedImmune , Phase 1 advanced solid tumors ( NCT01577745 ) , 60.

Techniques: Binding Assay