Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Generation and characterization of therapeutic anti-NOTCH3 mAbs (A) NOTCH3-dependent report gene assay with NOTCH3-targeted and control mAbs. Data represent mean ± SEM from 3 biological replicates (n = 4 per replicate), ns, non-significant. (B) qRT-PCR of HES1 gene expression in treated cells. Data represent mean ± SD from 2 biological replicates (n = 3 per replicate), ns, non-significant. (C) NOTCH3 immunoblot from mAb-treated cells and xenografts. GAPDH is shown as a loading control. Schematic diagram of NOTCH3 cleavage events and protein fragments. M, mouse number. (D and E) Immunoblot using N- or C-terminal domain antibodies to detect NOTCH3 fragments after DLL4 activation in treated cells. β-actin is shown as a loading control. (F) Epitope mapping of anti-NOTCH3 mAbs using NRR3-NRR1 domain swap chimeric constructs. NRR3 domains are shown in black and NRR1 domains are shown in gray. Representative data represent mean (n = 2). (G) Binding interface of anti-N3(i) Fab on the NOTCH3-NRR domain as determined from the co-crystal structure is shown in red, LNR-A (green), LNR-B (beige), LNR-C (blue), HD1 (pink), and HD2 (magenta).

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Gene Assay, Control, Quantitative RT-PCR, Gene Expression, Western Blot, Activation Assay, Construct, Binding Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Generated, Activity Assay, In Vitro, Control, Knockdown, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Control, Polymer, Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Adjuvant, Antibody Labeling, RNAscope, Sequencing, Software

Journal: Microcirculation (New York, N.y. : 1994)

Article Title: EHD2 modulates Dll4 endocytosis during blood vessel development

doi: 10.1111/micc.12740

Figure Lengend Snippet: Membranous Dll4 localizes with EHD2 and caveolin‐1. (A) Representative image of proximity ligation assay (PLA). Cells were stained as indicated and PLA reaction is marked by red puncta. Control condition was not transfected. Dll4‐myc condition was only transfected with Dll4‐myc and stained for endogenous EHD2. (B) Graph of number of PLA puncta by condition. Control condition was not transfected. EHBP1 condition was transfected with EHBP1‐HA and Dll4‐myc. Dll4‐myc condition was only transfected with Dll4‐myc and stained for endogenous EHD2. (C) Schematic of engineered Dll4 (top). Bottom, cartoon of pH‐dependent function of GFP variant pHluorin tag. PHluorin fluoresces on the membrane at neutral pH but is quenched when internalized into acidic endosomes allowing for visualization of only membranous Dll4. (D) Representative image of endothelial cell (HUVEC) stained for caveolin‐1 (Cav1) expressing pHluorin‐Dll4 (pH‐Dll4) and RFP‐EHD2. (E) Representative image of cell stained for actin expressing pH‐Dll4 and RFP‐EHD2. (F) Representative image of cell stained for clathrin expressing pH‐Dll4 and RFP‐EHD2. (G) Proportion of coincidence of clathrin (y‐axis) and EHD2 (x‐axis) around Dll4 puncta. (H) Pearson's correlation between indicated proteins. N, number of cells. Boxes denote magnified images on right. Yellow arrowheads show areas of pH‐Dll4 puncta. *** p < .001. Error bars are 95% confidence intervals. All experiments were done at minimum in triplicate

Article Snippet: Dll4 was purchased from Origene (MR212151).

Techniques: Proximity Ligation Assay, Staining, Control, Transfection, Variant Assay, Membrane, Expressing

Journal: Microcirculation (New York, N.y. : 1994)

Article Title: EHD2 modulates Dll4 endocytosis during blood vessel development

doi: 10.1111/micc.12740

Figure Lengend Snippet: EHD2 and caveolae localize to adherens junctions in sprouts. (A) Schematic of 3‐dimensional sprout growth in fibrin‐bead assay (FBA) using Human umbilical vein endothelial cells. (B) Representative sprout stained for VE‐cadherin (VE‐Cad) expressing pHluorin‐Dll4 (pH‐Dll4). Yellow line marks line scan area. (C) Line scan of pH‐Dll4 and VE‐Cad of image in panel B. (D) Representative sprout expressing pH‐Dll4 and red fluorescence protein (RFP)‐EHD2. Yellow line marks line scan area. (E) Line scan of pH‐Dll4 and RFP‐EHD2 of image in panel D. (F) Representative sprout stained for VE‐Cad expressing RFP‐EHD2. Yellow line marks line scan area. (G) Line scan of pH‐Dll4 and VE‐Cad of image in panel F. (H) Representative image of retinal blood vessels in P6 mouse stained for isolectin, Dll4, and caveolin‐1 (Cav1). Boxes are magnified images on right. Yellow line marks line scan area. (I) Line scan of Cav1 and Dll4 in panel H. Yellow arrowheads show areas of Dll4 puncta. All experiments were done at minimum in triplicate. All images are a single confocal slice

Article Snippet: Dll4 was purchased from Origene (MR212151).

Techniques: Staining, Expressing, Fluorescence

Journal: Microcirculation (New York, N.y. : 1994)

Article Title: EHD2 modulates Dll4 endocytosis during blood vessel development

doi: 10.1111/micc.12740

Figure Lengend Snippet: Loss of EHD2 blunts Dll4 endocytosis. (A) Western blot of EHD2 after siRNA (si) treatment in comparison to scramble (Scram) control in Human vein endothelial cells. (B) Schematic of pHrodo‐labeled delta‐like ligand 4 (Dll4) antibody. PHrodo gains fluorescent intensity with increasing endosomal pH, thus used as a metric of endocytosis. (C) Relative internalization of pHrodo‐Dll4 pulse‐chase over time between indicated siRNA groups. IgG was used as a non‐specific internalization control. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): Scram si vs. EHD2 si; Scram IgG vs. pHrodo‐Dll4 with EHD2 si. (D) Relative internalization of pHrodo‐Dll4 pulse‐chase over time between indicated groups. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): DMSO vs. Dynosore; DMSO vs. MβCD. (E) Schematic of recombinant Notch intracellular domain (NECD) functionalized to microbead and labeled with pHrodo. (F) Relative internalization of functionalized NECD pulse‐chase over time between indicated siRNA groups. A minimum of 50 cells were used per group. Comparisons: Scram si vs. EHD2 si treated cells. (G) Relative internalization of functionalized NECD pulse‐chase over time between indicated siRNA groups. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): Scram si vs. Dll4 si; Scram si vs. Jagged1 (Jag1) si; Scram si vs. Dll4 and Jag1 si treated cells. (H) Relative internalization of functionalized NECD pulse‐chase over time between indicated siRNA groups. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): Scram si vs. caveolin1 (Cav1) si; Scram si vs. clathrin light chain B (CLTB) si; Scram si vs. AP2 Associated Kinase 1 (AAK1) si treated cells. (I) Antibody feeding assay comparing internalized to surface‐bound ratio Dll4 between indicated groups. N, number of cells. (J) Western blot of Dll4 levels across indicated treatment groups. (K) Relative expression of Hes1 compared to GAPDH control in indicated si treatment groups. N, number of replicates. * p < .05, ** p < .01, *** p < .001, **** p < .0001. ns, non‐significant. Error bars are SEM. All experiments were done at minimum in triplicate

Article Snippet: Dll4 was purchased from Origene (MR212151).

Techniques: Western Blot, Comparison, Control, Labeling, Pulse Chase, Recombinant, Feeding Assay, Expressing

Journal: Cell Reports Methods

Article Title: Generation of dual-attribute iTNK cells from hPSCs for cancer immunotherapy

doi: 10.1016/j.crmeth.2024.100843

Figure Lengend Snippet:

Article Snippet: The human DLL4 gene (hDLL4) (NM_019074) (OriGene, Cat: SC113239) was cloned into the pCDH-MCS-T2A-Puro-MSCV Cloning and Expression Lentivector (System Biosciences, Cat: CD522A-1) and packaged to obtain lentiviral hDLL4 vector, in accordance with a previously described protocol.

Techniques: Staining, Purification, Recombinant, Modification, Membrane, Cell Stimulation, Wright Stain, Software