The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro
- Medizinische Hochschule Hannover, Germany
- Harvard Medical School, Massachusetts
- Dana Farber Cancer Institute, Massachusetts
Figures
Figure 1 with 3 supplements
Schematic representation of DLL1 and DLL4 and variant proteins.
I-X, full-length and chimeric ligands generated by domain swaps. XI and XII, ligands with exchanges of the known NOTCH1 contact amino acids in the MNNL and DSL domains. XIII, DLL4 variant with an …
Figure 1—figure supplement 1
Amino acid exchanges of DLL variant proteins.
(A) Amino acid sequences around the break points of chimeric DLL1 and DLL4 proteins. Amino acids unique to DLL1 and DLL4 are indicated in red and blue, respectively. Break point are just before the …
Figure 1—figure supplement 2
Analysis of ligand receptor binding.
(A) Analysis of the binding of various ligands to a biotinylated human N1 ectodomain fragment containing EGF repeats 6–15 using biolayer interferometry. Binding of DLL4 (from the N-terminus through …
Figure 1—figure supplement 3
N109 is highly conserved and N-glycosylated in DLL4.
(A) Alignment of murine DLL1 and DLL4 and of mammalian, bird, amphibian, and fish DLL4 showing the conserved N-glycosylation consensus site (green Asp in black box) in DLL4 proteins. M.m., Mus …
Figure 2
The extracellular domains of DLL1 and DLL4 determine ligand behavior during somitogenesis.
(A) Scheme of the targeting vector pMP8.CAG-Stop used to introduce inducible chimeric ligands into the Hprt locus, and of Cre-mediated activation of transgene (D1ECD_D4ICD or D4ECD_D1ICD) expression …
Figure 3
D1N-E3_D4 is not able to compensate for DLL1 function during somitogenesis.
(A) "Mini-gene“ targeting strategy to express DLL1 or DLL4 variants from the Dll1 locus (a) and alleles generated in this study (d and e). The Dll1Dll1ki (b) and Dll1Dll4ki (c) control alleles were …
Figure 4 with 1 supplement
DLL1 and DLL4 differentially activate NOTCH1 and NOTCH2 in cell-based co-culture assays.
(A) RT-PCR analysis using RNA of HeLaN1 cells shows the expression of endogenous human NOTCH2 and NOTCH3 in addition to the exogenous murine Notch1. (B) ES cell-based trans-activation assays …
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Figure 4—source data 1
Raw data used to generate the graph in Figure 4B.
Relative luciferase activity (units) of different assays are means of ≥2 technical replicates (measurements of the same cell lysate) of each (co-)culture.
- https://doi.org/10.7554/eLife.40045.013
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Figure 4—source data 2
Data used to generate the graphs in Figure 4C and Figure 4—figure supplement 1A.
DLL1 and DLL4 protein expression level in ES cells determined by quantitative analysis of Western blots (DLL1 or DLL4 expression intensity/𝛽-Tubulin intensity (DLL/𝛽-Tub)). Expression of DLL4 clone #1 was normalized to expression of DLL1 clone #1 analyzed in the same assay. The value obtained in Assay #7 (red) represents an outlier (determined by GraphPad Prism7) and was not included in the calculation of the average.
- https://doi.org/10.7554/eLife.40045.014
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Figure 4—source data 3
Raw data used to generate the graph in Figure 4D.
Relative cell surface levels of DLL1 (ES clone #1) and DLL4 (ES cell clone #1) proteins determined by cell surface biotinylation and quantitative analysis of Western blots after immunoprecipitation.
- https://doi.org/10.7554/eLife.40045.015
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Figure 4—source data 4
Numerical values used to generate the graphs in Figure 4E.
Relative luciferase activity (units) for each assay was calculated by subtraction of E14 background values. Normalized activation (fold change) was obtained by normalization to DLL1 activity and correction for protein and cell surface levels based on the values for relative protein expression (Figure 4—source data 2) and cell surface presentation (Figure 4—source data 3). Normalized activation = normalized activation x [prot level DLL1/prot level DLL4] x [rel surface level DLL1/rel surface level DLL4].
- https://doi.org/10.7554/eLife.40045.016
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Figure 4—source data 5
Numerical values used to generate the graphs in Figure 4F.
Luciferase activity (units) for each assay was calculated by subtraction of the E14 background values. Normalized activation (fold change) was obtained by normalization to DLL1 activity and correction for protein and cell surface levels based on the values for relative protein expression (Figure 4—source data 2) and cell surface presentation (Figure 4—source data 3). Normalized activation = normalized activation x [prot level DLL1/prot level DLL4] x [rel surface level DLL1/rel surface level DLL4].
- https://doi.org/10.7554/eLife.40045.017
Figure 4—figure supplement 1
Consistent N1 and N2 activation by different cell clones expressing DLL4.
(A) Protein expression level in DLL1 clone #1 and DLL4 clone #1 (DLL ligand/β-Tub) determined by quantitative Western blot analysis. Each dot represents a technical replicate (numerical values in Fig…
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Figure 4—figure supplement 1—source Data 1
Numerical values used to generate the graph in Figure 4—figure supplement 1B.
DLL1 and DLL4 protein levels in different ES cell clones were determined by quantitative analysis of Western blots and the relative protein expression was obtained by normalization to DLL1 clone #1 analyzed in the same assay. The value obtained in assay 13 (red) represents an outlier (determined by ROUT analysis using GraphPad Prism7) and was not included in the calculation of the average.
- https://doi.org/10.7554/eLife.40045.011
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Figure 4—figure supplement 1—source Data 2
Numerical values used to generate the graphs in Figure 4—figure supplement 1C,D.
N1 and N2 activation by different ES cell clones expressing DLL1 and DLL4.
- https://doi.org/10.7554/eLife.40045.012
Figure 5 with 1 supplement
Contributions of the MNNL-EGF3 portion and contact amino acids to ligand selectivity towards N1 and N2.
(A) N1/N2 activation ratios by DLL1 and DLL4 chimeric proteins show that receptor selectivity of DLL1 and DLL4 is encoded by the extracellular domain and that EGF3 contributes to N1/N2 selectivity. …
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Figure 5—source data 1
Raw data (RLUs) of luciferase activity in co-cultures with N1rep cells used to generate the graph in Figure 5—figure supplement 1A.
Values represent relative luciferase activity (units) after subtraction of E14 background RLUs.
- https://doi.org/10.7554/eLife.40045.020
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Figure 5—source data 2
Raw data (RLUs) of luciferase activity in co-cultures with N1rep cells used to generate the graph in Figure 5—figure supplement 1B.
Values represent relative luciferase activity (units) after subtraction of E14 background RLUs.
- https://doi.org/10.7554/eLife.40045.021
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Figure 5—source data 3
N1/N2 activation ratios.
Values represent N1/N2 activation ratio. Values were used for generation of graphs in Figure 5A and D. Red values were identified as outliers (determined by ROUT analysis by GraphPad Prism7) and excluded from calculations.
- https://doi.org/10.7554/eLife.40045.022
Figure 5—figure supplement 1
N1 and N2 activation by different ligand proteins.
(A) and (B) Rel. luciferase activity obtained in co-cultures of ES cells expressing wild type ligands, chimeric ligands, and ligands with amino acid substitutions in the direct ligand-receptor …
Figure 6
DLL1 carrying the DLL4 contact amino acids in the MNNL and DSL domains is a functional DLL1 ligand in vivo.
(A) E15.5 Dll1D1contD4ki/D1contD4ki (c; n = 12) fetuses are indistinguishable from wild type (a; n = 19) and Dll1Dll1ki/Dll1ki (b; n = 3) controls. (B) D1contD4 co-localizes with pan-Cadherin …
Author response image 1
Expression of D1N-E3_D4 cannot be quantified with confidence due to a closely co-migrating background band detected by the anti-Flag antibody.
(A) Expression of wild type DLL1 and DLL4 and of chimeric ligand proteins. Protein migration size varies due to differential post-translational modifications of the extracellular domains of DLL1 and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | DLL1 | MGI:104659; NCBI Gene: 13388 | ||
| Gene (Mus musculus) | DLL4 | MGI:1859388; NCBI Gene: 54485 | ||
| Strain, strain background (Mus musculus) | CD1 | Charles River Laboratories | ||
| Strain, strain background (Mus musculus) | 129Sv/CD1 hybrids | own colony | ||
| Genetic reagent (Mus musculus) | Dll1lacZ | PMID: 9109488; DOI: 10.1038/ 386717a0 | RRID:MGI:5780046 | |
| Genetic reagent (Mus musculus) | Dll1loxP | PMID: 15146182; DOI: 10.1038/ ni1075 | RRID:MGI:5431505 | |
| Genetic reagent (Mus musculus) | T(s):Cre | PMID: 18708576; PMCID: PMC2518812; DOI: 10.1101/gad. 480408 | MGI:3811072 | |
| Genetic reagent (Mus musculus) | ZP3:Cre | PMID: 10686600 | MGI:2176187 | |
| Genetic reagent (Mus musculus) | Dll1Dll1ki | PMID: 26801181; PMCID: PMC4788113; DOI: 10.1534/genetics. 115.184515 | MGI:5790945 | |
| Genetic reagent (Mus musculus) | Dll1Dll4ki | PMID: 26114479; PMCID: PMC4482573; DOI: 10.1371/journal. pgen.1005328 | MGI:5779556 | |
| Genetic reagent (Mus musculus) | Dll1D1N-E3_D4ki | this paper | mini gene insertion in the Dll1 locus | |
| Genetic reagent (Mus musculus) | Dll1D1contD4ki | this paper | mini gene insertion in the Dll1 locus | |
| Genetic reagent (Mus musculus) | HprtDll1ECD_Dll4ICD | this paper | inducible insertion into Hprt locus | |
| Genetic reagent (Mus musculus) | HprtDll4ECD_Dll1ICD | this paper | inducible insertion into Hprt locus | |
| Cell line (Mus musculus) | E14TG2a | PMID: 26114479; PMCID: PMC4482573; DOI: 10.1371/journal. pgen.1005328 | ||
| Cell line (Mus musculus) | 129Sv/cast | PMID: 26114479; PMCID: PMC4482573; DOI: 10.1371/journal. pgen.1005328 | ||
| Cell line (Homo sapiens) | HeLaN1 | PMID: 9653148; DOI: 10.1073/pnas. 95.14.8108 | ||
| Cell line (Mus musculus) | pMP8.CAG-DLL1 | PMID: 26801181; PMCID: PMC4788113; DOI: 10.1534/genetics. 115.184515 | ||
| Cell line (Mus musculus) | E14rep | PMID: 26801181; PMCID: PMC4788113; DOI: 10.1534/genetics. 115.184515 | ||
| Cell line (Mus musculus) | N1rep | PMID: 26801181; PMCID: PMC4788113; DOI: 10.1534/genetics. 115.184515 | ||
| Cell line (Mus musculus) | N2rep | PMID: 26801181; PMCID: PMC4788113; DOI: 10.1534/genetics. 115.184515 | ||
| Bacterial strain (E. coli) | SW106 | PMID:15731329 | ||
| Transfected construct (Mus musculus) | pMP8.CAG-DLL4 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG-D1ECD _D4ICD | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG-D1N- E3_D4 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG-D1N- E2_D4 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG- D1N-D_D4 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG- D4ECD_D1ICD | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG- D4N-E3_D1 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG- D4N-E2_D1 | this paper | progenitor: pMP8.CAG | |
Transfected construct (Mus musculus) | pMP8.CAG- D4N-D_D1 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG- D1contD4 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG- D4contD1 | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG- D4N109G | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG-Stop -D1ECD_D4ICD | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | pMP8.CAG-Stop- D4ECD_D1ICD | this paper | progenitor: pMP8.CAG | |
| Transfected construct (Mus musculus) | D1N-E3_D4-targeting | this paper | based on Dll1Dll1ki targeting | |
| Transfected construct (Mus musculus) | D1contD4-targeting | this paper | based on Dll1Dll1ki targeting | |
| Transfected construct (Mus musculus) | pLexM-Avi-His | this paper | progenitor: pLexM | |
| Transfected construct (Mus musculus) | pLexM-D1N-E5-Avi-His | this paper | progenitor: pLexM | |
| Transfected construct (Mus musculus) | pLexM-D4N-E5-Avi-His | this paper | progenitor: pLexM | |
| Transfected construct (Mus musculus) | pLexM-D1N-E3_D4-E5-Avi-His | this paper | progenitor: pLexM | |
| Transfected construct (Mus musculus) | pLexM-D4N-E3_D1-E5-Avi-His | this paper | progenitor: pLexM | |
| Transfected construct (Mus musculus) | pLexM-D1contD4-E5-Avi-His | this paper | progenitor: pLexM | |
| Transfected construct (Mus musculus) | pLexM-D4contD1-E5-Avi-His | this paper | progenitor: pLexM | |
| Transfected construct (Mus musculus) | pLexM-D4N109G-E5-Avi-His | this paper | progenitor: pLexM | |
| Antibody | Rat anti-DLL1 | PMID: 17664336; PMCID: PMC2064846; DOI: 10.1083/jcb. 200702009 | (1F9, rat monoclonal) | 1:50 (IF) |
| Antibody | Goat anti-DLL4 | R and D Systems | Cat. #AF1389 RRID:AB_354770 | 1:50 (IF) |
| Antibody | Mouse anti- panCadherin | Sigma-Aldrich | Cat. #C1821 RRID:AB_476826 | 1:250 (IF) |
| Antibody | Donkey anti- mouse Alexa 555 | Invitrogen | Cat. #A-31570 RRID:AB_2536180 | 1:100 (IF) |
| Antibody | Donkey anti-goat Alexa 488 | Invitrogen | Cat. #A-11055 RRID:AB_2534102 | 1:100 (IF) |
| Antibody | Donkey anti-rat Alexa 488 | Invitrogen | Cat. #A-21208 RRID:AB_2535794 | 1:100 (IF) |
| Antibody | Anti-FLAG- Peroxidase (HRP) | Sigma-Aldrich | (M2 mouse, monoclonal purified) Cat. #A8592 RRID:AB_439702 | 1:10 000 (WB) |
| Antibody | Mouse anti-β-Tubulin | Sigma-Aldrich | Cat. #T7816 RRID:AB_261770 | 1:500 000; 1:1 000 000 (WB) |
| Antibody | Anti-mouse HRP | Amersham | Cat. #NA931 RRID:AB_772210 | 1:10 000 (WB) |
| Antibody | MHC (Myosin Heavy Chain) | Sigma-Aldrich | Cat. #M4276 RRID:AB_477190 | 1:250 (IHC) |
| Antibody | Anti-DIG AP fab fragment | Roche | Cat. #1093274 | 1: 5 000 (ISH) |
| Antibody | Anti-mouse biotinylated (BA9200/goat) | Vector Laboratories | Cat. #BA-9200 RRID:AB_2336171 | 1:200 (IHC) |
| Commercial assay or kit | Luciferase Cell Culture Lysis 5X Reagent | Promega | Cat. #E1531 | |
| Commercial assay or kit | Luciferase Assay Reagent | Promega | Cat. #E1483 | |
| Commercial assay or kit | SuperScript IV Reverse Transcriptase | Invitrogen | Cat. #18090050 | |
| Commercial assay or kit | Expand High-Fidelity PCR system | Roche | Cat. #04743733001 | |
| Commercial assay or kit | Tri-Reagent | Sigma-Aldrich | Cat. #T9424 | |
| Chemical compound, drug | Sulfo-NHS-LC-Biotin | Thermo | Cat. #21335 | |
| Chemical compound, drug | Pierce NeutrAvidin Agarose | Thermo | Cat. #29200 | |
| Chemical compound, drug | cOmplete, Mini, EDTA-free Proteinase Inhibitor Cocktail | Roche | Cat. #04693159001 | |
| Chemical compound, drug | BM-Purple AP substrate Roche | Sigma-Aldrich | Cat. #11442074001 | |
| Chemical compound, drug | G418 | Biochrom | Cat. #291–25 | 125 μg/ml |
| Chemical compound, drug | HAT | Gibco | Cat. #31062–037 | 1:300 |
| Chemical compound, drug | HT | Gibco | Cat. #11067030 | 1:100 |
| Chemical compound, drug | Tunicamycin | Sigma-Aldrich | Cat. #T7765 | 1 μg/ml |
| Chemical compound, drug | Alcian blue | Sigma-Aldrich | Cat. #A5268 | 5% working solution |
| Chemical compound, drug | Alizarin red | Sigma-Aldrich | Cat. #A5533 | 5% working solution |
| Other | WesternBright Quantum HRP substrate | Advansta | Cat. #12042-D20 | as recommended by the manufacturer |
| Other | Amersham ECL Detection Reagent | GE Healthcare Life Sciences | Cat. #RPN2106 | as recommended by the manufacturer |
| Sequence-based reagent | DLL1 wt For | other | NA | 5‘-CTGAAGCGACCT GGCCCTGATAGCAC-3’ |
| Sequence-based reagent | DLL1 wt Rev | other | NA | 5‘-GGAGCTCCAGA CCTGCGCGGG-3’ |
| Sequence-based reagent | Dll1lacZ For | other | NA | 5‘-ATCCCTGGGT CTTTGAAGAAG-3’ |
| Sequence-based reagent | Dll1lacZ Rev | other | NA | 5‘-TGTGAGCGAGTA ACAACCCGTCGGATT-3’ |
| Sequence-based reagent | Dll1Dll1ki For | other | NA | 5‘-GGTTTGGGGAT CCATAACTTCG-3’ |
| Sequence-based reagent | Dll1Dll1ki Rev | other | NA | 5‘-GCCAGTCAGTTC CCAGTAAGAAGTC-3’ |
| Sequence-based reagent | Dll1Dll4ki For | other | NA | 5‘-AAGGACAACC TAATCCCTGCCG-3’ |
| Sequence-based reagent | Dll1Dll4ki Rev | other | NA | 5‘-TGCCACATCG CTTCCATCTTAC-3’ |
| Sequence-based reagent | Dll1loxP For | other | NA | 5‘-GCATTTCTCAC ACACCTC-3’ |
| Sequence-based reagent | Dll1loxP Rev | other | NA | 5‘-GAGAGTACTT GATGGAGCAAG-3’ |
| Sequence-based reagent | T(s):Cre For | other | NA | 5‘-AATCTTTGG GCTCCGCAGAG-3’ |
| Sequence-based reagent | T(s):Cre Rev | other | NA | 5‘-ACGTTCACCGGC ATCAACG-3’ |
| Sequence-based reagent | ZP3:Cre For | other | NA | 5‘-GCCTGCATTACC GGTCGATGCAACGA-3’ |
| Sequence-based reagent | ZP3:Cre Rev | other | NA | 5‘-GTGGCAGATGGC GCGGCAACACCATT-3’ |
| Sequence-based reagent | Hprt-CAGD1ECD_ D4ICD + neo For | this paper | NA | 5‘-CCTAGCCCCTGCA AGAACGGAGC-3’ |
| Sequence-based reagent | Hprt-CAGD1ECD_ D4ICD + neo Rev | this paper | NA | 5‘-TTGCCACAATTG GACTTGTC-3’ |
| Sequence-based reagent | Hprt-CAGD4ECD_ D1ICD + neo For | this paper | NA | 5‘-CACTGTGAGCAT AGTACC TTGAC-3’ |
| Sequence-based reagent | Hprt-CAGD4ECD_ D1ICD + neo Rev | this paper | NA | 5‘-CATGGTTTCTGTCT CTCCCCCACAGGG-3’ |
| Sequence-based reagent | HprtD1ECD_D4ICDrec and HprtD4ECD_D1ICDrec For (activated allele) | this paper | NA | 5‘-ACATGGCCGTCATC AAAGAG-3’ |
| Sequence-based reagent | HprtD1ECD_D4ICDrec and HprtD4ECD_D1ICDrec Rev (activated allele) | this paper | NA | 5‘-GGGCAACAGAGA AATATCCTGTCTC-3’ |
| Sequence-based reagent | Dll1D1N-E3_D4ki For | this paper | NA | 5‘-CTGTCTGCCAGG GTGTGATGACCAAC-3’ |
| Sequence-based reagent | Dll1D1N-E3_D4ki Rev | this paper | NA | 5‘-ATCGCTGATG TGCAGTTCACA-3’ |
| Sequence-based reagent | Dll1D1N-E3_D4ki For | this paper | NA | 5‘-TGCAGGAG TTCGTCAACAAG-3’ |
| Sequence-based reagent | Dll1D1N-E3_D4ki Rev | this paper | NA | 5‘-ATAGTGGCC AAAGTGGTCATC CCGAGGCTT-3’ |
| Sequence-based reagent | Y-Chromosome For | other | NA | 5‘-CTGGAGCTCT ACAGTGATGA-3’ |
| Sequence-based reagent | Y-Chromosome Rev | other | NA | 5‘-CAGTTACCAA TCAACACATCAC-3’ |
| Sequence-based reagent | mNotch1 For | other | NA | 5‘-TAGGTGCTC TTGCGTCACTTGG-3’ |
| Sequence-based reagent | mNotch1 Rev | other | NA | 5‘-TCTCCCCACT CGTTCTGATTGTC-3’ |
| Sequence-based reagent | hNOTCH1 For | PMID: 22002304; DOI: 10.1038/onc .2011.467 | NA | 5‘-TCCACCAG TTTGAATGGTCA-3’ |
| Sequence-based reagent | hNOTCH1 Rev | PMID: 22002304; DOI: 10.1038/onc. 2011.467 | NA | 5‘-AGCTCATCA TCTGGGACAGG-3’ |
| Sequence-based reagent | hNOTCH2 For | this paper | NA | 5‘-CAACCGCCA GTGTGTTCAAG-3’ |
| Sequence-based reagent | hNOTCH2 Rev | this paper | NA | 5‘-GAGCCATG CTTACGCTTTCG-3’ |
| Sequence-based reagent | hNOTCH3 For | PMID: 16327489; PMCID: PMC1409885 | NA | 5‘-AGATTCTCA TCCGAAACCGCTCTA-3’ |
| Sequence-based reagent | hNOTCH3 Rev | PMID: 16327489; PMCID: PMC1409885 | NA | 5‘-GGGGTCTC CTCCTTGCTATCCTG-3’ |
| Sequence-based reagent | hGAPDH For | PMID: 22002304; DOI: 10.1038/onc. 2011.467 | NA | 5‘-GAGTCAACG GATTTGGTCGT-3’ |
| Sequence-based reagent | hGAPDH Rev | PMID: 22002304; DOI: 10.1038/onc. 2011.467 | NA | 5‘-TTGATTTTGG AGGGATCTCG-3’ |
| Sequence-based reagent | Forward primer - correct integration into Hprt locus | other | NA | 5’-GGGAACCTGTT AGAAAAAAAGA AACTATGAAGAAC-3’ |
| Sequence-based reagent | Reverse primer - correct integration into Hprt locus | other | NA | 5’-GGCTATGAACTAATG GACCCCG-3’ |
| Sequence-based reagent | Forward primer - correct integration into Dll1 locus | other | NA | 5‘-TGTCACGT CCTGCACGACG-3’ |
| Sequence-based reagent | Reverse primer - correct integration into Dll1 locus | other | NA | 5‘-GGTATCGGA TGCACTCATCGC-3’ |
| Sequence-based reagent | guideA-For | this work, according to http://crispr.mit.edu/ | NA | 5'-GGCAGCGGG CAGCTCCGGAT-3' |
| Sequence-based reagent | guideB-Rev | this work, according to http://crispr.mit.edu/ | NA | 5'-GCTCTCGGG GTCGTCGCTGC-3' |
| Recombinant DNA reagent | Uncx-probe (plasmid) | DOI 10.1007/ s004270050120 | ||
| Recombinant DNA reagent | pLexM (plasmid) | DOI 10.1074/ jbc.M113.454850 | ||
| Recombinant DNA reagent | Cas9 D10A nickase (plasmid) | DOI 10.1126/ science.1231143 | Addgene #42335 | |
| Recombinant DNA reagent | Dll1 5' SB probe | PMID: 26801181; PMCID: PMC4788113; DOI: 10.1534/genetics. 115.184515 | 5’ probe: a 316 bp BamHI/AvaII fragment 3.8 kb upstream of Dll1 exon 1 | |
| Recombinant DNA reagent | Dll1 3' SB probe | PMID: 26801181; PMCID: PMC4788113; DOI: 10.1534/genetics. 115.184515 | 3’ probe: a 528 bp PCR fragment in Dll1 intron five obtained with primers CCTGTGAGACTTTCTA CGTTGCTC/CACAACCATGTCA CCTTCTAGATTC | |
| Software, algorithm | ImageJ; FIJI | RRID:SCR_003070 | ISAC Manager | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | Olympus | Olympus FLUOVI EW FV1000 | RRID:SCR_014215 |
Additional files
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Supplementary file 1
Relative cell surface expression levels of the ligand proteins used co-culture studies.
Levels of one representative clone for each ligand protein were determined by cell surface biotinylation and quantitative analysis of Western blots after immunoprecipitation. Values for DLL1 and DLL4 see Figure 4—source data 3. ND: due to closely co-migrating background band protein levels could not be quantified. Surface expression validated by biotinylation of ES cells and antibody staining of PSMs.
- https://doi.org/10.7554/eLife.40045.024
-
Supplementary file 2
Relative ligand protein expression level in ES cell clones.
The protein level of three independent clones used for co-culture studies was determined by quantitative analysis of Western blots and normalized to DLL1 clone #1 protein level measured in the same assay. Values for DLL1 and DLL4 see Figure 4—source data 2. ND: due to closely co-migrating background band protein levels could not be quantified.
- https://doi.org/10.7554/eLife.40045.025
-
Transparent reporting form
- https://doi.org/10.7554/eLife.40045.026
