Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) C2C12 cells were engineered to expressed Notch1 receptors lacking the extracellular domain (N1DECD, green). This receptor is inactive in the presence of the γ-secretase inhibitor DAPT (red), but constitutively active when DAPT concentration is reduced in the culture medium. (B) Comparison of transcript levels in C2C12-N1ΔECD cells at 1 hr or 6 hr after DAPT removal. The blue line represents equal expression at 1 hr and 6 hr, and the gray lines represent 5-fold changes in either direction. Circled genes are putative direct Notch targets. The blue circle highlights target genes that are upregulated >5-fold at 1 hr but not 6 hr, while red circles indicate target genes that are upregulated >5-fold only after 6 hr. See also and . (C) qPCR time course measurement of Hes1 (blue), Hey1 (orange), and HeyL (yellow) mRNA levels following complete DAPT removal at t = 0 hr. (D) Duration dependence of Hes1 (blue) and Hey1 (orange) response to DAPT removal for 5 min, 15 min, or 30 min followed by replenishment (“Pulse”), or no replenishment until the 1 hr or 4 hr measurement (“Sustained”). Error bars represent SEM calculated from duplicate experiments (n = 2). See also Figure S4 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Concentration Assay, Comparison, Expressing

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Both Dll1 (blue) and Dll4 (red) activate the Notch1 receptor (green) to induce proteolytic release of the Notch intracellular domain (NICD), but are used in different biological contexts (blue and red boxes, bottom). The released NICD translocates to the nucleus and, in complex with CSL/RBPjκ (yellow), activates Notch target genes (white). (B) Left: Engineered CHO-K1 “sender” cell lines contain stably integrated constructs expressing Dll1 (blue) or Dll4 (red), each with a co-translational (T2A, brown) H2B-mCh readout (purple), from a 4epi-Tetracycline (4epi-Tc) inducible promoter. Right: “Receiver” cells stably express a chimeric receptor combining the Notch1 extracellular domain (Notch1ECD) with a Gal4 transcription factor (orange), which can activate a stably integrated fluorescent H2B-3xCitrine reporter gene (chartreuse). (C) Left (schematics): A minority of receiver cells (green) are co-cultured with an excess of either Dll1 (blue) or Dll4 (red) sender cells. Right: Filmstrips showing representative sustained (top, Dll4 senders) or pulsatile (bottom, Dll1 senders) response of a single receiver cell (center, automatically segmented nucleus outlined in white). Grey channel shows DIC images of cells, while the rate of increase in Citrine fluorescence, scaled to 25%–75% of its total range, is indicated using green pseudo-coloring. See also and . (D) Left: Representative traces showing total nuclear Citrine fluorescence levels (top) or corresponding derivatives of the total Citrine ( d Citrine/ d t), i.e., promoter activity (bottom), in individual receiver cells activated by Dll4. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll4. Solid traces represent medians, lighter shades indicate SEM, and gray shading indicates SD. n, number of traces included in the alignment. See for alignment and normalization procedure. (E) Left: Corresponding plots (as in D) showing total nuclear Citrine fluorescence levels (top) and promoter activity (bottom) in individual receiver cells in co-culture with Dll1. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll1. The percentage value (60%) in the plots on right indicates the fraction of receiver traces included in the alignment ( STAR Methods , see also ). (F) 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in the traces included in (D) and (E). This time window is chosen to simultaneously estimate the promoter activity at the peak of Dll1 pulses and at steady-state levels of Dll4 signaling. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided Kolmogorov-Smirnov (K-S) test. See also Figures S1 and S2 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Stable Transfection, Construct, Expressing, Cell Culture, Fluorescence, Activity Assay, Co-Culture Assay

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Developing chick embryo (dorsal view schematic). Dll1 (blue cells in 3) is expressed in a fraction of neural crest cells (gray, see 2, 3). These cells activate Notch1-expressing Pax7 + progenitor cells in the dorsomedial lip (DML, magenta) of the somite. When activated, these progenitor cells (green, 3) upregulate Hes1 and the muscle regulatory gene MyoD1. (B–D) Representative images showing effects of Dll1 or Dll4 electroporation into the neural crest, on Hes1, Hey1, and MyoD1 expression in the DML. White arrows indicate the somites on the electroporated side. The dotted lines indicate the DMLs of somites or the central line of the neural tube. (B) Top: Dll1-T2A-EGFP (i, blue), electroporated into the left side of the neural tube, is expressed in the neural tube and neural crest, resulting in upregulation of Hes1 (ii, red) and MyoD1 (iii, green) in the somites on the electroporated (left) side compared to the right side, which serves as negative control. Bottom: When Dll4-T2A-EGFP (iv, blue) is electroporated, Hey1 (v, red) is upregulated on the electroporated side, and MyoD1 (vi, green) expression is decreased. (C) Dll1-T2A-EGFP (blue, left) electroporation does not affect expression of Hey1 (red, right) in adjacent somites. (D) Dll4-T2A-EGFP (blue, left) electroporation increases expression of Hes1 (red, right) in adjacent somites. See also and .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Expressing, Electroporation, Negative Control

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A and B) Dll4 ECD -Dll1 ICD and Dll1 ECD -Dll4 ICD were constructed by exchanging the intracellular domain (ICD) of Dll4 with that of Dll1. (A) Median response profiles in activated receiver cells co-cultured with Dll4 sender cells (red, top left) or Dll4 ECD -Dll1 ICD sender cells (magenta, right) under excess receiver conditions (as in ). Solid traces represent medians, lighter colored regions represent SEM, and gray shading represents SD. n, number of cell traces included in the alignment. See for alignment and normalization procedures. Bottom left: 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in individual traces included in the averaging. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided K-S test. (B) Corresponding response profiles (right, top left) and amplitudes (bottom left) in activated receiver cells co-cultured with Dll1 sender cells (blue) or Dll1 ECD- Dll4 ICD sender cells (purple) under excess sender conditions. (C) Representative images of “excess sender” co-cultures of receiver cells (R) expressing full-length Notch1 and sender cells (S) expressing either Dll4 ECD -Dll1 ICD (left) or Dll4 (Dll4 ECD -Dll4 ICD , right), immunostained for Notch1ECD. Examples of dispersed, low intensity staining or higher-intensity puncta are indicated by the white circles. (D) Left: Median values of number of puncta detected (see ) in Dll1 ICD (blue) or Dll4 ICD (red) sender cells neighboring receiver cells. Right: Median values of the (background subtracted) mean pixel intensity of dispersed signal (see ) within Dll1 ICD (blue) or Dll4 ICD (red) sender cells that neighbor receiver cells. Error bars represent SEM. p value calculated using the two-sided K-S test. (E) Schematic: Proposed differences in the abilities of ligands containing the Dll1 (blue) and Dll4 (red) ICDs to initiate transendocytosis in different clustering states. See also Figure S6 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Construct, Cell Culture, Activity Assay, Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL

doi: 10.1084/jem.20161715

Figure Lengend Snippet: A13 Notch3 agonist antibody activates a ligand-insensitive Notch3 receptor with the C455R mutation in vitro. (A) Schematic diagram showing the structure of the Notch3 receptor and the binding site where the A13 antibody interacts with the NRR domain. Lin12-Notch (LNR), heterodimerization domain (HD), and transmembrane domain (TM) are shown. Luciferase reporter assay for Notch signaling in monocultures of HEK 293 cells expressing Notch3 WT receptor (B) or the Notch3 C455R (C) after treatment with A13 or control IgG antibodies. TET indicates concentration of tetracycline used to induce transgene expression. Luciferase reporter assay for Notch signaling in HEK 293 cell co-cultures. HEK 293 cells expressing Notch3 WT or C455R Notch3 receptors in co-culture with control cells (D) or with HEK 293 cells expressing Jagged 1 (E). HEK 293 cells expressing Notch3 WT in co-culture with control cells (F) or with cells expressing Jagged 1 (G) treated with IgG or A13 antibodies. HEK 293 cells expressing Notch3 C455R in co-culture with control cells (H) or with cells expressing Jagged 1 (I) treated with IgG or A13. ELISA measurements of N3ECD in cell culture supernatant for each culture, Notch3 WT (J) and C455R (K). Luciferase reporter assay for Notch signaling in monocultures of HEK 293 cells expressing Notch3 WT receptor in the presence of compound E (L). ELISA measurements of N3ECD in cell culture supernatant in the presence of compound E (M). Luciferase reporter assay for Notch signaling in monocultures of HEK cells expressing Notch3 WT receptor treated with increasing concentrations of A13 (N). ELISA measurements of N3ECD in cell culture supernatant from cells treated with increasing concentrations of A13 (O). Notch3 signaling activation measured via gene reporter assay correlated with increased levels of N3ECD in cell culture supernatants measured by ELISA (P). (B–P) *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were analyzed via ANOVA. Values in graphs are expressed as the means ± SEM. The results are representative of three independent experiments each including three technical replicates per condition.

Article Snippet: The recombinant human N3ECD, originally used to raise the antibodies (1559-NT-050; R&D Systems), was used as the standard, and a positive control.

Techniques: Mutagenesis, In Vitro, Binding Assay, Luciferase, Reporter Assay, Expressing, Control, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL

doi: 10.1084/jem.20161715

Figure Lengend Snippet: A13 Notch3 agonist antibody reverses plasma biomarker changes in N3ECD and endostatin/collagen 18α1 in CADASIL mice. Dot plots showing plasma levels of N3ECD (A), endostatin/collagen 18α1 (B), IGFBP-1 (C), and HTRA1 (D) in 6-wk-old C455R mice treated with the A13 ( n = 10) or IgG ( n = 8) antibodies from 10 independent rounds of injections. (A-D) **, P < 0.01. Statistical analysis was done via unpaired two-tailed Student's t test. Each data point represents the value for a single mouse. Horizontal lines show the means ± SEM. The C455R mice were littermates.

Article Snippet: The recombinant human N3ECD, originally used to raise the antibodies (1559-NT-050; R&D Systems), was used as the standard, and a positive control.

Techniques: Clinical Proteomics, Biomarker Discovery, Two Tailed Test

Journal: eLife

Article Title: Cis- activation in the Notch signaling pathway

doi: 10.7554/eLife.37880

Figure Lengend Snippet: ( A ) Schematic of actual and potential cis - and trans- interaction modes in the Notch pathway. ( B ) Schematic of the N1D1 + Rfng cell line. CHO-K1 cells were engineered to express a chimeric receptor combining the Notch1 extracellular domain (‘N1ECD’, green) with the Gal4 transcription factor (orange) in place of the endogenous intracellular domain. When activated, released Gal4 activates a stably integrated fluorescent H2B-Citrine reporter gene (yellow) through UAS sites (brown) on the promoter. Cells also contain a stably integrated construct expressing Dll1 (red) with a co-translational (2A, white) H2B-mCherry readout (‘mCH’, red), from a 4-epiTc-inducible promoter. Cells also constitutively express Rfng (purple) and H2B-Cerulean (‘H2B-Cer’, blue). ( C ) ( Left ) Schematic of cis- activation assay conditions. A minority of N1D1 + Rfng (blue nuclei) cells were mixed with an excess of wild-type CHO-K1 cells (white nuclei). The typical distance between N1D1 + Rfng cells is ∆x. ( Right ) Filmstrip showing activation (Citrine fluorescence, green) of an isolated N1D1 + Rfng cell using time-lapse microscopy. Constitutive cerulean fluorescence (blue) in the same cell nucleus is also shown (see for additional examples). ( D ) Peak Notch activation rate in isolated N1D1 + Rfng cells (y-axis) versus distance to each of its closest neighboring N1D1 + Rfng cell (x-axis) at the point of maximum activity. One cell width is indicated by gray shaded area. Solid blue line indicates linear fit, whose flat slope suggests a cell-autonomous, distance-independent process. ( E ) Box plots showing the distribution of peak Notch activation rates in isolated N1D1 + Rfng cells prior to the first cell division in the cis- activation assay, for three different median Dll1 induction levels (indicated by numbers below bars; see for corresponding distributions).

Article Snippet: Cells were then incubated with either 10 ug/ml mouse IgG 2A control protein or 10 ug/ml mouse Notch1 Fc chimera protein (R and D Systems, Minneapolis, MN) along with DAPT and 4-epiTc overnight at 37°C, 5% CO 2 .

Techniques: Stable Transfection, Construct, Expressing, Activation Assay, Fluorescence, Isolation, Time-lapse Microscopy, Activity Assay

Journal: eLife

Article Title: Cis- activation in the Notch signaling pathway

doi: 10.7554/eLife.37880

Figure Lengend Snippet: ( A ) ( Left ) The N1 WT D1+Rfng cell line (schematic). CHO-K1 cells were engineered to express wild-type Notch1 receptor (‘N1 WT ’, green), an H2B-Citrine reporter (yellow) activated by cleaved NICD through 12 multimerized CSL binding sites in the promoter region (orange), and a Dll1-mCherry protein (red), from a 4-epiTc inducible promoter. Constructs for constitutive expression of Rfng (purple) and H2B-Cerulean (‘H2B-Cer’, blue) were also stably integrated. ( Right ) Flow cytometry analysis of the mean activation of N1 WT D1+Rfng cells in the cis -activation assay. The non-monotonic dependence of activation on ligand levels qualitatively resembles that observed for N1D1 + Rfng cells . ( B ) ( Left ) Expression levels of Notch receptors, ligands, and Fringes in wild-type NMuMG cells, measured using RNAseq. (Middle) Western blot analysis of endogenous Notch2 and Jagged1 after CRISPR-Cas9 mediated knockout in NMuMG cells. Notch2 and Jagged1 are visible in wild-type (WT) cells but absent in knockout cells (ΔN2ΔJ1). ( Right ) Activation level of WT NMuMG N1ECD-Gal4 (N1) and ΔN2ΔJ1 NMuMG N1ECD-Gal4 (N1-ΔN2ΔJ1) receiver cells by co-culture with NMuMG Dll1 sender cells. An equal number of Dll1 cells were cultured with either N1 or N1-ΔN2ΔJ1 receiver cells and analyzed for Notch activation (Citrine levels) by flow cytometry after 48 hr of co-culture. ( C ) Schematic of the NMuMG N1D1 + Rfng cell line. NMUMG ΔN2ΔJ1 cells were engineered to express a chimeric receptor combining the Notch1 extracellular domain (‘N1ECD’, green) with the Gal4 transcription factor (orange) in place of the endogenous intracellular domain, and fused to the Ankyrin domain of the Notch1ICD (ANK, dark blue). When activated, Gal4-ANK is released and enables activation of a stably integrated fluorescent H2B-Citrine reporter gene (yellow) through UAS sites (brown) on the promoter. A Dll1 (red) with co-translational (T2A, white) H2B-mCherry readout (red), expressed from a Tet-off promoter was also stably integrated. A constitutively expressed rTetR-HDAC4 (‘rTetS’) gene (pink) suppresses expression of the Dll1-T2A-H2B-mCherry cassette in the presence of doxycyline (‘Dox’). Rfng (purple) is expressed co-translationally with rTetS. Cells also constitutively express H2B-Cerulean (‘H2B-Cer’, blue). ( D ) Representative images showing surface staining (green) of N1ECD-Gal4 (top, left ) or N1ECD-Gal4-ANK (top, right ) receptors in NMuMG cells (gray overlay shows DIC channel). Notch receptor accumulated baso-laterally and was not observed on apical surfaces in the absence of the ANK domain. Inclusion of the ANK domain restored apical localization (white arrow). Bottom plot shows activation of N1ECD-Gal4 receiver (No ANK) and N1ECD-Gal4-ANK receiver (ANK) cells by co-cultured Dll1 senders. Inclusion of the ANK domain increased Notch activation (Citrine expression). Control cells are Notch receiver cells plated without Dll1 sender cells. ( E ) Wild-type (top) and engineered (bottom) Notch receptor show similar staining patterns in non-polarized CHO-K1 cells, resembling polarized NMuMG N1ECD-Gal4-ANK cells (D, upper right). ( F ) Filmstrip showing activation of an isolated NMuMG N1D1 + Rfng cell using time-lapse microscopy in reporter (green) and cerulean (blue) channels (See for additional examples). ( G ) ( Left ) Mean relative Notch activation (Citrine reporter fluorescence) vs. relative Dll1 expression levels (measured using the co-translational mCherry fluorescent protein) in NMuMG N1D1 + Rfng cells in the cis- activation assay. Values are normalized to Citrine and mCherry expression in untreated cells. Dll1 expression was varied by treating cells with 0, 1, or 10 µg/ml doxycycline or by transfecting in additional Dll1 (Materials and methods). Data represent the mean values across three replicate experiments, and error bars represent s.e.m. ( Right ) Expression level of ectopic Dll1 in NMuMG N1D1 + Rfng cells (untreated, equivalent to value of 1 in plot on left ) compared to endogenous Jag1 expression in wild-type NMuMG cells, measured by RNA-seq.

Article Snippet: Cells were then incubated with either 10 ug/ml mouse IgG 2A control protein or 10 ug/ml mouse Notch1 Fc chimera protein (R and D Systems, Minneapolis, MN) along with DAPT and 4-epiTc overnight at 37°C, 5% CO 2 .

Techniques: Binding Assay, Construct, Expressing, Stable Transfection, Flow Cytometry, Activation Assay, Western Blot, CRISPR, Knock-Out, Co-Culture Assay, Cell Culture, Staining, Control, Isolation, Time-lapse Microscopy, Fluorescence, RNA Sequencing

Journal: eLife

Article Title: Cis- activation in the Notch signaling pathway

doi: 10.7554/eLife.37880

Figure Lengend Snippet: ( A ) ( Top ) Cell lines used for analyzing effect of Rfng (purple) on cis -activation, in the context of Notch1 and Dll4. ( Bottom ) Comparison of mean cis -activation in polyclonal N1D4 cells with (purple, ‘N1D4 Pop + Rfng’) or without (black, ‘N1D4 Pop’) expression of Rfng, as a function of ligand expression (measured using fluorescence of the co-translated H2B-mCherry protein). Values represent mean of 3 replicate experiments, and error bars indicate s.e.m.

Article Snippet: Cells were then incubated with either 10 ug/ml mouse IgG 2A control protein or 10 ug/ml mouse Notch1 Fc chimera protein (R and D Systems, Minneapolis, MN) along with DAPT and 4-epiTc overnight at 37°C, 5% CO 2 .

Techniques: Activation Assay, Comparison, Expressing, Fluorescence

Journal: eLife

Article Title: Cis- activation in the Notch signaling pathway

doi: 10.7554/eLife.37880

Figure Lengend Snippet: ( A ) (Left) Schematic showing how recombinant N1ECD-Fc protein (rN1ECD-Fc) affects trans -activation between cells expressing ligands (red) and receptors (green). rN1ECD-Fc protein, present in excess, binds to surface ligands and prevents their interactions with receptors on neighboring cells. This is expected to reduce overall Notch activation in cells relative to the control (cells incubated with IgG protein). (Right) Comparison of mean Notch activation in N1D1+Rfng cells incubated with rNotch1ECD-Fc receptors (magenta) and those that were incubated with IgG (black) for 24 hr (see Materials and methods). Cells were plated densely to allow trans -activation and analyzed by flow cytometry <24 hr post-plating. ( B ) (Left) Schematic of N1D1+Rfng cells co-cultured with CHO-K1 cells (top) or Notch1 cells (‘N1-only cells’) that express receptor but no ligand. Notch1 cells can bind to the Dll1 ligand on the N1D1+Rfng cells and block it from interacting with Notch1 on the same cell. If surface interactions are necessary for cis -activation, this will decrease Notch activation. (Right) Comparison of mean Notch activation in N1D1+Rfng cells co-cultured with an excess (95% of culture) of CHO-K1 cells (black) or N1-only cells (magenta). ( C ) Mean Notch activation in N1D1+Rfng cells co-cultured at low (mostly cis -activation, ‘ Cis -only’) or high ( cis + trans- activation, ‘ Cis + trans ’) relative density with CHO-K1 cells and treated with 10 μM Blebbistatin (Materials and methods). Data is shown as fold-change relative to control untreated samples plated similarly. ( D ) Compared to adherent culture (blue), suspension culture (magenta) of N1D1+Rfng cells results in a slight decrease in cell-surface levels of mean Dll1 (left) and Notch1 (right), measured by immunostaining (see Materials and methods). In all panels, error bars represent s.e.m (n=3 replicate experiments).

Article Snippet: Cells were then incubated with either 10 ug/ml mouse IgG 2A control protein or 10 ug/ml mouse Notch1 Fc chimera protein (R and D Systems, Minneapolis, MN) along with DAPT and 4-epiTc overnight at 37°C, 5% CO 2 .

Techniques: Recombinant, Activation Assay, Expressing, Control, Incubation, Comparison, Flow Cytometry, Cell Culture, Blocking Assay, Suspension, Immunostaining

Journal: eLife

Article Title: Cis- activation in the Notch signaling pathway

doi: 10.7554/eLife.37880

Figure Lengend Snippet: ( A ) ( Top ) Cell lines used for analyzing effect of Rfng on cis -activation. ( Bottom ) Plots showing mean Notch activation (reporter Citrine fluorescence normalized to background fluorescence in uninduced cells) in N1D1 (black) or N1D1 + Rfng (purple) cells expressing different levels of Dll1 (measured using co-translational mCherry fluorescence). Error bars indicate s.e.m (n = 3 replicate experiments). ( B ) Heatmaps of mean Notch activation (n = 3 replicates), relative to background reporter fluorescence, in N1D1 + Rfng (upper panel) or N1D1 (lower panel) cells induced with different [4epi-Tc] (columns) and cultured at different relative fractions (rows). Upper panel is the same data in , replotted for direct comparison. Rfng expression predominantly affects signal amplitude (compare intensity scales). ( C,D ) ( Top ) Cell lines used for analyzing effect of ligand on cis -activation of Notch1 ( C ) or Notch2 ( D ). ( Bottom ) Comparison of mean cis- activation in polyclonal populations (‘Pop’) of cells co-expressing Dll1 or the higher affinity ligand Dll4 with the indicated receptor, as a function of ligand expression, read out by co-translated H2B-mCherry fluorescence. Values represent mean of 3 replicates. Error bars indicate s.e.m. Note difference in y-axis scales between panels C and D.

Article Snippet: Cells were then incubated with either 10 ug/ml mouse IgG 2A control protein or 10 ug/ml mouse Notch1 Fc chimera protein (R and D Systems, Minneapolis, MN) along with DAPT and 4-epiTc overnight at 37°C, 5% CO 2 .

Techniques: Activation Assay, Fluorescence, Expressing, Cell Culture, Comparison

Journal: eLife

Article Title: Cis- activation in the Notch signaling pathway

doi: 10.7554/eLife.37880

Figure Lengend Snippet:

Article Snippet: Cells were then incubated with either 10 ug/ml mouse IgG 2A control protein or 10 ug/ml mouse Notch1 Fc chimera protein (R and D Systems, Minneapolis, MN) along with DAPT and 4-epiTc overnight at 37°C, 5% CO 2 .

Techniques: Derivative Assay, Expressing, Transfection, Construct, Recombinant, Clone Assay, CRISPR, Plasmid Preparation, Sequencing, Knockdown, Negative Control, Control, Purification, RNA Extraction, cDNA Synthesis, SYBR Green Assay, Software

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 3. Mib1 and endocytosis of Jagged1 are required for activation of, but not binding to, Notch receptors. (A,B)12XCSL-luciferease reporter activity in full-length Notch1-expressing cells co-cultured with cells transiently transfected with (A) EGFP, Jagged1+EGFP or Jagged1+EGFP-tagged DynaminII K44A as indicated or (B) control siRNA, Jagged1+control siRNA or Jagged1+siRNA specific for MIB1 as indicated. Bar graphs show luciferase activity (arithmetic mean) from triplicates of each co-culture. Luciferase activity was normalized to control cultures transfected with EGFP (A) or control siRNA (B). Error bars indicate s.d.; **P<0.01. (C)Immunocytochemistry demonstrating that cells transfected with DynaminII K44A and Jagged1 (top cell) can bind N1ECD-Fc-Cy3, whereas cells not transfected with DynaminII K44A and Jagged1 (the bottom cell) does not bind N1ECD-Fc-Cy3. As a control for block of endocytosis by DynaminII K44A, there is no transferrin uptake in the top (DynaminII K44A expressing) cell, whereas transferrin is internalized in the bottom cell. Scale bar: 10m. Immunofluorescence images were adjusted using the levels function in Photoshop. In each case, adjustments were applied to the entire image. (D)Detection of ligand-receptor interaction by flow cytometry in cells transiently transfected with empty plasmid and EGFP, Jagged1 and EGFP or DynaminII K44A and EGFP, as indicated. (E)Detection of ligand-receptor interaction by flow cytometry in cells transiently transfected with empty plasmid and control siRNA, Jagged1 and control siRNA or Jagged1 and MIB1 siRNA, as indicated. Percentages of fluorescently labeled cells (arithmetic mean) is shown (bar graphs). Error bars indicate s.d.; *P<0.05, **P<0.01.

Article Snippet: Recombinant proteins and pharmacological inhibitors Recombinant rat Notch1 extracellular domain 1-12 fused to the human Fc chain of IgG1 (R&D Systems) was used essentially as described (Ladi et al., 2005).

Techniques: Activation Assay, Binding Assay, Activity Assay, Expressing, Cell Culture, Transfection, Control, Luciferase, Co-Culture Assay, Immunocytochemistry, Blocking Assay, Immunofluorescence, Flow Cytometry, Plasmid Preparation, Labeling

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 4. Analysis of ligand and receptor localization following ligand-receptor interaction. (A)Immunocytochemistry of co-cultured HEK293T cells expressing Jagged1 and Notch1. In a cell with ongoing Notch signaling, as determined by a fluorescent 12XCSL-EGFP reporter of Notch signaling (bottom cell), Notch immunoreactivity (C-terminal) was seen in the nucleus and intracellular vesicles (in more than 95% of the cells), whereas Notch immunoreactivity was more predominant at the cell surface in a cell not engaged in active Notch signaling (cell in top-right corner). Jagged1 immunoreactivity (C-terminal) was found in intracellular vesicles in the ligand-expressing cell (middle cell), which is interfaced with the cell with active Notch signaling (bottom cell). Scale bar: 10m. (B)HEK293 cells stably expressing Jagged1WT or Jagged1Ndr were co-cultured with HEK293T cells transfected with Notch1 and 12xCSL-dsRed. Notch1- expressing cells adjacent to Jagged1WT-expressing cells displayed red fluorescence and nuclear Notch1 C-terminus staining (arrowheads). Scale bar: 50m. (C)Quantification of dsRed- and Notch1-positive cells shows that significantly more Notch1-positive cells adjacent to Jagged1WT cells manifest red fluorescence compared with cells not adjacent to Jagged1WT cells in the same culture or Notch1-positive cells adjacent to Jagged1Ndr cells.

Article Snippet: Recombinant proteins and pharmacological inhibitors Recombinant rat Notch1 extracellular domain 1-12 fused to the human Fc chain of IgG1 (R&D Systems) was used essentially as described (Ladi et al., 2005).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Stable Transfection, Transfection, Fluorescence, Staining

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 6. Jagged1 is ubiquitylated by Mind bomb in response to Notch interaction. HEK293T or HEK293T cells expressing Notch1 were co-cultured with HEK293 cells or HEK293 cells expressing Jagged1WT or Jagged1Ndr

Article Snippet: Recombinant proteins and pharmacological inhibitors Recombinant rat Notch1 extracellular domain 1-12 fused to the human Fc chain of IgG1 (R&D Systems) was used essentially as described (Ladi et al., 2005).

Techniques: Expressing, Cell Culture

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 7. NECD is transendocytosed into Jagged1WT-expressing cells and transported through the endocytic degradation pathway. (A)HEK293T cells expressing full length HA-Notch1 (N-terminally tagged Notch1) were co-cultured with Jagged1WT-expressing HEK293T cells transfected with EGFP-EEA1. HA immunoreactivity was observed in EGFP-positive and Jagged1-positive vesicles indicating trans-endocytosis of Notch1 ECD into EEA1-positive vesicles in Jagged1-expressing cells. Boxed region is magnified in inset. Scale bar: 20m. (B)Jagged1WT cells exhibit high levels of trans-endocytosis of Notch1 ECD, whereas Jagged1Ndr cells exhibit low-to-no trans-endocytosis of Notch1ECD into Jagged1- and EEA1-positive vesicles (arrowheads). Scale bar: 20m. (C-E)Immunocytochemistry of cells with fluorescently labeled subcellular markers and incubated with N1ECD-Fc-Cy3. (C)Immediately after incubation, N1ECD-Fc-Cy3 (red) colocalized with the plasma-membrane marker EGFP-F. (D)At 1 hour, N1ECD-Fc-Cy3 (red) was detected in EGFP-EEA1-positive vesicles. (E)At 4 hours, N1ECD-Fc-Cy5 (green) was detected in lysosomes, as visualized by the lysosome marker Lysotracker Red (red).

Article Snippet: Recombinant proteins and pharmacological inhibitors Recombinant rat Notch1 extracellular domain 1-12 fused to the human Fc chain of IgG1 (R&D Systems) was used essentially as described (Ladi et al., 2005).

Techniques: Expressing, Cell Culture, Transfection, Immunocytochemistry, Labeling, Incubation, Clinical Proteomics, Membrane, Marker