recombinant human dll4  (R&D Systems)

Journal: The FEBS journal

Article Title: Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

doi: 10.1111/febs.16946

Figure Lengend Snippet: Fig. 2. Loss of Dmt1 results in diminished endogenous Notch1 signalling, which is rescued by overexpression of Nicd. (A) mRNA expression levels of Notch target genes, Hey1 and Hey2, in Dmt1 WT and KO MEFs stimulated with Dll4 and treated with DMSO or GSI for 24 h, measured by qRT-PCR. Csnk2a2 mRNA expression was employed as a housekeeping control [one-way ANOVA (Tukey compari- son), *P < 0.05, **P < 0.01, ***P < 0.001, significantly compared with Dmt1 WT cells stimulated with Dll4]. (B) Fluorescent ectopic expres- sion of Nicd1-mCherryGFP in Dmt1 WT and KO cells upon doxycycline (DOX)-induction. Cells were counterstained with DAPI. Scale bar: 10 lm. GSI, c-secretase inhibitor dibenzazepine. Data are representative of three independent experiments, and values are expressed in mean SD.

Article Snippet: Delta-like 4 (Dll4) stimulation was performed using recombinant human Dll4 (R&D Systems, Minneapolis, MN, USA; cat. #1506-D4-050/CF).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control

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: bioRxiv

Article Title: Bmp9 regulates Notch signaling and the temporal dynamics of angiogenesis via Lunatic Fringe

doi: 10.1101/2023.09.25.557123

Figure Lengend Snippet: Bmp9 upregulates LFng expression in endothelial cells. (A) Schematic representation of the crosstalk between Bmp9 and Vegf-Notch signaling in ECs, as well as of the simulation and in vitro experiments. Green and blue lines represent previously established characteristics of the crosstalk; in red, the hypothesized LFng upregulation by Bmp9 is reported. (B-E) Experiments (full color) and simulations (striped color) of qPCR analysis of Hes1, Hey1 and LFng in HUVECs, induced by Bmp9 injection and/or Dll4 coating after 24h stimulation, with (bright color) or without (shaded color) LFng siRNA treatment. Number of repeats was higher than 4 in all cases ((B, D) N=5; (E) N=4). (F) Western blot analysis of NICD and LFng compared to the safe-keeping gene beta-actin in HUVECs with control (left) or LFng siRNA treatment (right) upon stimulation with 10ng/ml Bmp9, up to 24h, with relative quantification normalized over unstimulated data (bottom). The number of independent repeats per condition was N = 3. (G) IsolectinB4 and LFng staining of wild type and Alk1ΔEC retinal vessels, with associated normalized quantification of qPCR analysis. For the qPCR analysis, the number of independent experimental repeats per condition was N = 3. Scale bar, 200 µm. All values are mean +/− SD. *p < 0.05; **p<0.01; ***p<0.001; ****p<0.0001; (B, D-E) ANOVA, (G) Unpaired t-test.

Article Snippet: Quantitect qPCR primers for Jag1 (QT00031948), Hey1 (QT00035644), Hey2 (QT00026971), Dll4 (QT00081004), Hes1 (QT00039648), LFng (QT00212240), MFng (QT00084007), and RFng (QT01160075) were purchased from Qiagen, Recombinant BMP9 (3209-BP-010) and Dll4 (1506-D4-050) were obtained from R&D systems.

Techniques: Expressing, In Vitro, Injection, Western Blot, Control, Quantitative Proteomics, Staining

Journal: bioRxiv

Article Title: Bmp9 regulates Notch signaling and the temporal dynamics of angiogenesis via Lunatic Fringe

doi: 10.1101/2023.09.25.557123

Figure Lengend Snippet: Bmp9-mediated LFng upregulation potentiates EC-pericyte interactions. (A) Schematic representation of the signaling crosstalk between ECs and pericytes, as well as of the simulation and in vitro experiments. Full names correspond to unbound ligands and receptors (e.g. Notch1), while capital letters with numbers correspond to bound receptors (e.g. N1.D4 is the bound Notch1-Dll4 complex). Straight and curved arrows represent upregulation and protein complex formation, respectively, while the ⊣ symbol represents inhibition. (B-G) Experiments (full color) and simulations (striped color) of qPCR analysis of Hes1 and Hey1 in HUVECs and pericytes, cultured alone or in co-culture, induced by Bmp9 injection (10 ng/ml) after 24h stimulation, with (bright color) or without (shaded color) Jag1 or LFng siRNA treatment. The number of independent experimental repeats was N = 5. (H) qPcR analysis of LFng and Jag1 in HUVECs induced by Bmp9 injection (10 ng/ml) after 24h stimulation, with or without LFng siRNA treatment. The number of independent experimental repeats was N = 7. (I) qPCR analysis of Jag1 in HUVECs stimulated by Bmp9 (10 ng/ml) and/or Dll4 (10 ng/ml) at different time points, up to 24h. The number of independent experimental repeats was N=7. All values are mean +/− SD. *p < 0.05; **p<0.01; ***p<0.001; ****p<0.0001; ANOVA.

Article Snippet: Quantitect qPCR primers for Jag1 (QT00031948), Hey1 (QT00035644), Hey2 (QT00026971), Dll4 (QT00081004), Hes1 (QT00039648), LFng (QT00212240), MFng (QT00084007), and RFng (QT01160075) were purchased from Qiagen, Recombinant BMP9 (3209-BP-010) and Dll4 (1506-D4-050) were obtained from R&D systems.

Techniques: In Vitro, Inhibition, Cell Culture, Co-Culture Assay, Injection