Journal: Nature cell biology

Article Title: Adherens junctions organize size-selective proteolytic hotspots critical for Notch signaling

doi: 10.1038/s41556-022-01031-6

Figure Lengend Snippet: (a) A schematic showing membrane compartmentalization choreographing the sequential steps in cell-surface activation of Notch. LRE and RIP represent distinct membrane microdomains (µdomains) for Notch receptor-ligand engagement and regulated intramembrane proteolysis, respectively. (top) A representative image showing LRE and RIP µ-domains. Scale bar, 2 µm. (b) Representative confocal fluorescence images showing Dll1, Notch1 (N1), and presenilin1 (PS1) distributions at the interfacial membrane formed between cells co-expressing Notch1 and Dll1 in the presence of TAPI2. The area with a white dashed line indicates the cellular interface. (left) A maximum projection image of Dll1 and Notch1 constituting a LRE µdomain.[to authors: to save word space in your figure legend, you may combine the several descriptions at the end. I have provided an example of the scale bars; please verify if correct]. (right) Individual fluorescent channel and merged images for Dll1, N1, and PS1.[to authors: to save word space in your figure legend, you may combine the several descriptions at the end. I have provided an example of the scale bars; please verify if correct].. (c) Manders’ overlap coefficients (MOCs) of Notch with Dll1, PS1 with N1, and PS1 with Dll1, respectively. n = 18 cells examined for each condition, pooled over three independent experiments. (d) A schematic of spatial distribution of Notch intermediates during the cell-surface activation pathway. (e) Representative confocal images of N1 and PS1 within the RIP µdomains from the cells activated by culturing them on a Dll4-Fc immobilized substrate with DAPT. (left) A maximum projection image of enriched Notch-mCherry signal at RIP-µdomains. . (Top-right) Magnified individual fluorescence channel images of the boxed region.. (right) Z-resliced images showing the sections of the cellular interfaces. Scale bar, 4 µm. (f) MOCs of Notch1 with PS1 during sequential molecular processing of Notch1. n = (left to right) 11, 12, and 10 cells examined over two independent experiments. (g) A schematic showing AJ-mediated membrane compartmentalization that creates LRE- and RIP-microdomains. (h) Representative confocal fluorescence images of RIP- (PS1) and LRE- (Dll1 and N1) µdomains relative to AJs. (left) A maximum projection image. Inset shows a magnified image of the boxed area highlighting the membrane microdomains at cellular interfaces.. (right) z-resliced images. (i) MOCs of PS1, Dll1, and Notch1 localization with E-cadherin. n = (left to right) 15, 11, and 14 cells examined over two independent experiments. (c, f, i) For box and whisker plots, colored centre lines and (+) marks indicate median and mean, respectively. The boxes show the 25th to 75th percentiles, and the whiskers extend to the minima and the maxima. ****P < 0.0001, ns: non-significant; one-way ordinary ANOVA followed by Tukey’s multiple comparison. Scale bars are (a) 2 µm, (b, left) 5 µm, (b,right) 2 µm, (e, left) 10 µm, (e top right and right) 4 µm, (h) 2 µm.

Article Snippet: Glass-bottomed dishes (MatTek, #1.5, D = 10 mm) were coated with recombinant human E-cadherin-Fc (50 µg/ml, R&D systems), recombinant human Dll4-Fc (2.5 µg/ml, Sino Biological), and fibronectin (5 µg/ml, Sino Biological) diluted in PBS for 1 hr at 37°C, and rinsed with 10 ml PBS with calcium and magnesium (UCSF cell culture facility).

Techniques: Membrane, Activation Assay, Fluorescence, Expressing, Whisker Assay, Comparison

Journal: Nature cell biology

Article Title: Adherens junctions organize size-selective proteolytic hotspots critical for Notch signaling

doi: 10.1038/s41556-022-01031-6

Figure Lengend Snippet: (a) A schematic to capture the spatial distribution of Notch intermediates during the cell-surface activation pathway. (b) Confocal z-resliced images showing Notch distribution (red) relative to AJ (green) from the cells without Dll4 activation (i), treated with Dll4 and TAPI2 (ii), treated with Dll4 and DAPT (iii), and washed out to remove DAPT inhibition (iv). Scale bar, 3 µm. (c) Quantification of Notch signal enrichment at the AJs during the activation. Notch enrichment (IIN/IOUT) is calculated as the ratio of average Notch fluorescence intensity within AJs (IIN) and outside AJ (IOUT). The enrichment factor of Dil is present as a control showing AJ-independent distribution. In the box-whisker plot, the boxes show the 25th to 75th percentiles, and the whiskers extend to the 10th and 90th percentiles, with individual data points above the whiskers shown for the lowest and highest 10% of each dataset. Solid lines and (+) marks indicate median and mean, respectively. n = (left to right) 13, 4, 25, 17 cells analyzed across three independent experiments. *** P = 0.0005, ****P < 0.0001, ns: non-significant, one-way ordinary ANOVA followed by Tukey’s multiple comparison testing. (d) Representative time-course confocal z-resliced images showing S2-cleaved Notch at AJs as a function of time after DAPT removal. The NICD signal (red) at the AJ gradually decreases, indicating NICD release. Images shown here are not from identical cells, but represent a general trend of NICD signal at AJs for each time point. Scale bar, 5 µm (e) Quantification IIN/IOUT ratio as a function of time after DAPT washout. Data are the mean ± s.d of n = 25 (+DAPT), 9 (0 hr), 10 (0.5 hr), 6 (1.5 hr), 8 (3 hr), 17 (12 hr), and 14 (-Dll4) biological replicates examined across 3 independent experiments.

Article Snippet: Glass-bottomed dishes (MatTek, #1.5, D = 10 mm) were coated with recombinant human E-cadherin-Fc (50 µg/ml, R&D systems), recombinant human Dll4-Fc (2.5 µg/ml, Sino Biological), and fibronectin (5 µg/ml, Sino Biological) diluted in PBS for 1 hr at 37°C, and rinsed with 10 ml PBS with calcium and magnesium (UCSF cell culture facility).

Techniques: Activation Assay, Inhibition, Fluorescence, Whisker Assay, Comparison

Journal: Nature cell biology

Article Title: Adherens junctions organize size-selective proteolytic hotspots critical for Notch signaling

doi: 10.1038/s41556-022-01031-6

Figure Lengend Snippet: (a) Representative epi-fluorescence images showing Notch activation in U2OS SNAP-NFL-Gal4 reporter cell lines in different cellular environments: Group of cells on a Dll4-Fc coated substrate (left), solitary cells with no prior contact on a Dll4-Fc coated substrate (middle), and solitary cells plated on a Dll4-Fc and Ecad-Fc coated substrate (right). Scale bars, 20 µm. (b) Representative low magnification epi-fluorescence image showing both grouped cells and multiple solitary cells. Scale bar, 100 µm. (c) Quantification of Notch activation by measuring H2B-mCherry fluorescence changes in cells within a group (n = 152 cells from 3 independent experiments), solitary cells (n = 50 cells from 3 independent experiments). ** P = 0.0034 (unpaired two-tailed Student’s t test). (d) Quantification of Notch activation in solitary cells cultured on a Dll4-Fc coated substrate and those cultured on a Dll4-Fc and Ecad-Fc coated substrate (n = 27 cells for both conditions from 3 independent experiments). ** P = 0.005 (unpaired two-tailed Student’s t test). (e) Representative confocal images of H2B-mCherry fluorescence in U2OS SNAP-NFL-Gal4 reporter cells (wt), E-cadherin knockout cells (Ecad-KO), Ecad-KO cells with recombinant E-cadherin transfection (Ecad-KO + Ecad), and Ecad-KO cells with N-cadherin transfection (Ecad-KO + Ncad). Cytosol labeled with CMFDA dye was shown for wt and Ecad-KO cells. E-cadherin and N-cadherin were shown for Ecad-KO + Ecad and Ecad-KO + Ncad cells. Scale bar, 100 µm. (f) Quantification of Notch activation in the wt (n = 86), Ecad-KO (n = 100), Ecad-KO + Ecad (n = 52), and Ecad-KO + Ncad (n = 80) cells (all pooled from 2 independent experiments). **** P < 0.0001 (ordinary one-way ANOVA followed by Tukey’s). (c, d, and f) Boxes and whiskers indicate the interquartile and full ranges, respectively. Black lines and (+) marks indicate median and mean, respectively. (g) Comparison of Notch signal activation, readout by mean nuclear H2B-mCherry fluorescence, as a function of E-cadherin expression, readout by membrane GFP fluorescence signal. Each dot represents H2B-mCherry signal of a single cell, and cells are grouped into bins based on their levels of Ecad expression. (from left to right) n = 94, 35, 71, 87, 50, 25, and 45 cells examined across two independent experiments. * P = 0.019, ** P = 0.049, *** P = 0.036, ns, non-significant (ordinary one-way ANOVA followed by Tukey’s). In the box-whisker plot, the red lines indicate median. The boxes and whiskers indicate the 25th to 75th percentiles, and the 10th to 90th percentiles, respectively.

Article Snippet: Glass-bottomed dishes (MatTek, #1.5, D = 10 mm) were coated with recombinant human E-cadherin-Fc (50 µg/ml, R&D systems), recombinant human Dll4-Fc (2.5 µg/ml, Sino Biological), and fibronectin (5 µg/ml, Sino Biological) diluted in PBS for 1 hr at 37°C, and rinsed with 10 ml PBS with calcium and magnesium (UCSF cell culture facility).

Techniques: Fluorescence, Activation Assay, Two Tailed Test, Cell Culture, Knock-Out, Recombinant, Transfection, Labeling, Comparison, Expressing, Membrane, Whisker Assay

Journal: British Journal of Cancer

Article Title: Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

doi: 10.1038/sj.bjc.6605368

Figure Lengend Snippet: Representative examples of immunohistochemistry in colon cancer showing membranous CA9 adjacent necrosis ( A ) and endothelial Dll4 ( B ) expression. Immunohistochemistry for Dll4 shows membranous and cytoplasmic endothelial expression in a colon adenoma ( C ) and epithelial expression associated with goblet cell differentiation in a neoplastic crypt (( D ) black arrows indicate Dll4-positive goblet cells, and red arrows indicate Dll4-negative non-goblet cells). Dll4 is also weakly expressed by neoplastic cells without goblet cell differentiation in a colon adenocarcinoma ( E ). Endothelial cells lining vessels (arrows) adjacent to normal colonic crypts did not express Dll4 by immunohistochemistry ( F ).

Article Snippet: Recombinant human Dll4 was cloned into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA).

Techniques: Immunohistochemistry, Expressing, Cell Differentiation

Journal: British Journal of Cancer

Article Title: Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

doi: 10.1038/sj.bjc.6605368

Figure Lengend Snippet: Statistical significance of associations between molecular variables

Article Snippet: Recombinant human Dll4 was cloned into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA).

Techniques:

Journal: British Journal of Cancer

Article Title: Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

doi: 10.1038/sj.bjc.6605368

Figure Lengend Snippet: Statistical significance of associations between molecular and categorical clinical variables

Article Snippet: Recombinant human Dll4 was cloned into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA).

Techniques:

Journal: British Journal of Cancer

Article Title: Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

doi: 10.1038/sj.bjc.6605368

Figure Lengend Snippet: Univariate analysis of overall survival

Article Snippet: Recombinant human Dll4 was cloned into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA).

Techniques:

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: RHOQ is induced by DLL4/Notch signalling in vitro. HUVECs were grown on BSA or rhDLL4-coated plates, harvested at indicated time points and changes in RHOQ and DLL4/Notch targets were assessed by a QPCR or b by western blotting. c Active RHOQ status following GTPase pull down assay and magnitude of active RHOQ was detected by western blotting. Changes in DLL4/Notch target expression following 16 h incubation with DBZ (20 nM) compared to DMSO equivalent controls was assessed by either. d QPCR, normalised to BSA DMSO control, or e by western blotting, or f cells were fixed and analysed for NICD bound to Notch promoter binding site for RHOQ, compared to that of DLL4 and HEY1 by chromatin immunoprecipitation. β-Actin as a loading control and densitometry was performed on western blots. Densitometry ratios were expressed relative to the first BSA control sample (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test comparing two data groups; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: In Vitro, Western Blot, Pull Down Assay, Expressing, Incubation, Binding Assay, Chromatin Immunoprecipitation

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ expression abolished expression of downstream DLL4/Notch signalling. HUVECs were either transfected with siRHOQ or infected with human RHOQ (hRHOQ); cells were then grown on BSA or rhDLL4-coated plates and harvested at indicated time points. Changes in DLL4/Notch downstream target expression in stimulated siRHOQ transfected HUVECs were assessed by a QPCR after 16 h stimulation, expressed relative to BSA control, and b western blotting, after either (i) 8 h or (ii) 24 h stimulation, using B-Actin as a loading control. c Changes in DLL4/Notch downstream target expression in stimulated HUVECs overexpressing hRHOQ was assessed over time by QPCR. Densitometry ratios were expressed relative to the first BSA control sample (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Expressing, Transfection, Infection, Western Blot

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Localisation changes of NICD in RHOQ negative cells. a Transfected HUVECs with RHOQ siRNA duplexes were cultured on BSA or rhDLL4 (1 μg/ml)-coated plates for 8 h for NICD analysis or 16 h for other markers before immuno-fluorescence staining for a RHOQ (green) and Exo70 (red), b NICD (green) with RHOQ (red) or Exo70 (red) and visualised by confocal microscopy. Nuclei stained with DAPI (blue). (Key: Scale bar = 20 nm; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Transfection, Cell Culture, Fluorescence, Staining, Confocal Microscopy

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: RHOQ/Exo70 co-localise with Notch components. a HUVECs were transfected with lentivirus to overexpress GFP tagged RHOQ and cultured on BSA or rhDLL4-coated plates and fixed 24 h later and Duo-link staining detecting only Exo70/NICD (red) associated antibodies, visualised by confocal microscopy. Nuclei stained with DAPI (blue). b Immunoprecipitation with either IgG control antibody, RHOQ antibody and Exo70 antibody and expression of Notch components assessed by western blotting, compared to whole cell (W) lysates, with B-Actin used as a loading control. (Key: White arrow, example of co-localisation; Scale bar = 20 nm; data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Transfection, Cell Culture, Staining, Confocal Microscopy, Immunoprecipitation, Expressing, Western Blot

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: NICD does not translocate to the nucleus in RHOQ negative cells. a Transfected HUVECs with RHOQ siRNA duplexes were cultured on BSA or rhDLL4 (1 μg/ml)-coated plates for 8 h before ( a ) cells were pelleted and lysed for whole cell lysate (W), or cytoplasmic fraction (C), nuclear fraction (N) and changes in the NICD localisation assessed by western blotting, using B-Actin and Lamin B as markers of fraction contamination and loading control or being fixed and b analysed for NICD binding to the Notch promoter binding site for DLL4 and Hey1 by chromatin immunoprecipitation (Data representative of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Transfection, Cell Culture, Western Blot, Binding Assay, Chromatin Immunoprecipitation

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ increases autophagy and leads to degradation of Notch1 receptor. HUVECs cultured on BSA or rhDLL4 (1 μg/ml)-coated plates and effects on autophagosomes were assessed 16 h later by staining cells with autophagy tracker (red) with changes in tracker levels, a visualised by confocal microscopy (nuclei stained with DAPI (blue)), and assessed by b FACs or c cells were fixed and immuno-fluorescence staining for localisation changes in RHOQ (red) or Notch1 (red) and LC3B (green) proteins and visualised by confocal microscopy (nuclei stained with DAPI (blue)) or d illustrating LC3B binding sites on Notch1 (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test between data group and control, Scale bar = 20 nm; representative images and data of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Cell Culture, Staining, Confocal Microscopy, Fluorescence, Binding Assay

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ expression leads to NICD degradation in lysosomes. HUVECs cultured on BSA or rhDLL4 (1 μg/ml)-coated plates and effects on lysosomes were assessed 16 h later by staining cells with lysosome tracker (red) with changes in tracker levels, a visualised by confocal microscopy (nuclei stained with DAPI (blue)) and assessed by b FACs or cells were fixed and immuno-fluorescence staining for localisation changes in proteins c duo-link staining detecting only Lamp/Notch1 (red) or Lamp1/NICD (green) associated antibodies, visualised by confocal microscopy. Nuclei stained with DAPI (blue). d Transfected HUVECs with RHOQ siRNA duplexes were cultured on BSA or rhDLL4 (1 μg/ml)-coated plates for 8 h with or without chloroquine (10 µM) before ( a ) changes in DLL4/Notch downstream target expression by QPCR (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test between data group and control, Scale bar = 20 nm; representative images and data of n = 3 independent experiments)

Article Snippet: HUVECs were seeded onto dishes pre-coated with BSA (1 μg/ml; Sigma-Aldrich) or rhDLL4 extracellular domain (1 μg/ml; R&D Systems, Minneapolis, USA) in 0.2% (w/v) gelatin (Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Staining, Confocal Microscopy, Fluorescence, Transfection

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Simultaneous binding to VEGF and Dll4 by HD105 bispecific antibody leads to effective blockade of VEGF/VEGFR2 and Dll4/Notch1 interactions. The HD105 bispecific antibody was constructed of the C-terminal of the anti-VEGF (bevacizumab-similar) IgG backbone linked with a single-chain Fv targeting Dll4 (A). The binding affinity of the HD105 bispecific antibody against human VEGF or human Dll4 was determined by Biacore assays (B) and ELISAs (C, D). The KD values of each antibody against VEGF or Dll4 are summarized in Table (B). The HD105 bispecific antibody (closed circle) dose-dependently bound to human VEGF (C) or Dll4 (D). In addition, the HD105 bispecific antibody simultaneously bound to each antigen, human VEGF and human Dll4, in dual-antigen capture ELISAs (E). The anti-Dll4 antibody (open circle in C) or the anti-VEGF (bevacizumab-similar) antibody (open circle in D, E) was used as negative control. Competitive ELISAs demonstrated that the HD105 bispecific antibody inhibited the interaction between VEGF/VEGFR2 (F) or Dll4/Notch1 (G) in a dose-dependent manner. The EC50 (half maximal effective concentration) values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) for VEGF/VEGFR2 inhibition were 2.98 ± 0.5 nM and 2.84 ± 0.41 nM, respectively (F). The EC50 values of the anti-Dll4 antibody (open circle) and HD105 bispecific antibody (closed circle) were 0.65 ± 0.06 nM and 1.14 ± 0.06 nM, respectively (G).

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Binding Assay, Construct, Negative Control, Concentration Assay, Inhibition

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Blockade of both VEGF/VEGFR2 and Dll4/Notch1 signaling pathways by HD105 bispecific antibody leads to inhibition of each signaling-induced cellular response. The HD105 bispecific antibody inhibited both the VEGF/VEGFR2 and the Dll4/Notch1 signaling pathways in HUVECs (A). The VEGF/VEGFR2 signaling pathway was monitored by the activation of VEGFR2 and ERK (phosphorylation). The Dll4/Notch1 signaling pathway was monitored by the generation of NICD (Notch-induced intracellular domain). HUVEC sprouting assays were performed in a fibrin gel in the presence of PBS (B), anti-VEGF (bevacizumab-similar) antibody (C), anti-Dll4 antibody (D), or HD105 bispecific antibody (E). Representative images show sprouting tip cells of HUVECs from the beads under basal media (B, arrowheads) and more sprouting under anti-Dll4 antibody treatment (D, arrows) but much less sprouting under anti-VEGF antibody (C) or HD105 bispecific antibody treatment (E). Scale bar (B-E), 150 μm. The bar graph (F) shows the measurement of sprouting HUVECs at 225 μm from beads (n = 20 beads/group, mean ± SE). *, P < 0.05 versus PBS. †, P < 0.05vs. anti-Dll4 antibody. The HD105 bispecific antibody inhibited VEGF-dependent HUVEC proliferation (G) and Dll4-induced Notch-1-dependent activation of luciferase in SKOV-3-RBP-J Κ luciferase cells (H) in a dose-dependent manner. The IC50 values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) on HUVEC proliferation were 1.49 ± 0.04 nM and 1.58 ± 0.08 nM, respectively (G). The IC50 values of the HD105 bispecific antibody (closed circle) and the anti-Dll4 antibody (open circle) on luciferase activation were determined to be 0.62 ± 0.23 nM and 0.58 ± 0.03 nM, respectively (H).

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Inhibition, Activation Assay, Luciferase

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Suppression of tumor progression in several cancer xenograft models by HD105 bispecific antibody. Human A549 lung cancer (A) or human SCH gastric cancer (B, C) was subcutaneously implanted into nude mice. After tumors were grown to an average volume of 150–200 mm3, PBS (open triangle), anti-VEGF (bevacizumab-similar) antibody (2.5 mg/kg, open circle), anti-mouse Dll4 antibody (2.5 mg/kg, closed triangle), or mouse HD105 bispecific antibody (3.25 mg/kg, closed circle) was intraperitoneally injected twice (A549) or once (SCH) per week (A, B). Tumor volume was calculated by the formula width2 × length × 0.52. The dose dependency of the mouse HD105 bispecific antibody was evaluated in human SCH gastric cancer xenograft model (C). PBS (open triangle) or mouse HD105 bispecific antibody (0.361 mg/kg, closed triangle; 1.083 mg/kg, open circle; 3.25 mg/kg, closed circle) was intraperitoneally injected once per week. The response to mouse HD105 bispecific antibody (6.5 mg/kg, once per week, closed circle) was also determined using other human gastric cancer xenograft models, including MKN-74 (D), SNU-5 (E), and SNU-16 (F). Tumor progression was not inhibited by the mouse HD105 bispecific antibody in MKN-74 and SNU-5 but was inhibited in SNU-16 similarly to SCH.

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Injection

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Suppression of tumor angiogenesis in cancer xenograft models by HD105 bispecific antibody. Fluorescence micrographs compare the vasculature of A549 human lung cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), or mouse HD105 bispecific antibody (D). Scale bar (A-D), 50 μm. The tumor vasculature was stained for CD31 immunoreactivity (green), and the vascular basement was stained for type IV collagen (red). Tumor vessels were decreased after treatment with anti-VEGF (bevacizumab-similar) antibody or mouse HD105 bispecific antibody, whereas tumor vessels were markedly increased after treatment with anti-mouse Dll4 antibody compared to PBS. Higher-resolution images compare the phenotype changes of tumor vessels in detail after PBS (E), anti-VEGF (bevacizumab-similar) antibody (F), anti-mouse Dll4 antibody (G), or mouse HD105 bispecific antibody treatment (H). Scale bar (E-H), 20 μm. The tumor vasculature was stained for CD31 immunoreactivity (red), and the perivascular pericyte was stained for NG2 (green). The nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole). Tumor vessels after treatment with anti-mouse Dll4 antibody were conspicuously thinner and more branched than the tumor vessels of other groups. Bar graph (I) measuring tumor vessel density of A549 tumor tissues in xenograft mice confirms the conspicuous increase of tumor vessels after anti-mouse Dll4 antibody treatment but decreases after anti-VEGF (bevacizumab-similar) antibody, mouse HD105 bispecific antibody, or combination treatment with anti-mouse Dll4 antibody and anti-VEGF (bevacizumab-similar) antibody. †, P < 0.05 versus PBS. *, P < 0.05vs. anti-Dll4 antibody. However, the functional tumor vessels in SCH gastric cancer tissues assessed by intravenous FITC-labeled Lycopersicon esculentum (Tomato) lectin staining were significantly decreased after treatment with anti-VEGF (bevacizumab-similar) antibody as well as anti-mouse Dll4 antibody (J). †, P < 0.05 versus PBS. ‡, < 0.05vs. anti-VEGF (bevacizumab-similar) antibody. *, P < 0.05 versus anti-Dll4 antibody. Functional tumor vessels were more decreased after treatment with mouse HD105 bispecific antibody compared to the other groups.

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Fluorescence, Staining, Functional Assay, Labeling

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Increase in apoptotic tumor cells in cancer xenograft models treated with HD105 bispecific antibody. Fluorescence micrographs show apoptotic cells stained for activated caspase-3 antibody (red) in SCH human gastric cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), and mouse HD105 bispecific antibody (D and E). Scale bar (A-D), 50 μm; (E), 20 μm. Nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole, blue). The higher-resolution image confirms that activated caspase-3 antibody was stained in the cytoplasm of the apoptotic cells after mouse HD105 bispecific antibody treatment (E). The bar graph (F) measuring the cell density of apoptotic cells in SCH cancer tissues confirms the significant increase in apoptotic cells after mouse HD105 bispecific antibody treatment. *, P < 0.05vs. PBS. ‡, < 0.05 versus anti-VEGF (bevacizumab-similar)) antibody. *, P < 0.05vs. anti-Dll4 antibody.

Article Snippet: Recombinant human Dll4 (100 ng/well) was coated onto white 96-well plates (Costar) for 24 hours at 4°C.

Techniques: Fluorescence, Staining