(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 …
(A) Schematic of ‘control’ cis-activation assay used to verify that the relative density of cells was low enough to prevent trans-interactions. N1D1 + Rfng cells lacking H2B-Cerulean expression …
(A) Histograms of mCherry fluorescence in cells analyzed in Figure 1E. Cells were categorized as expressing low, medium, or high Dll1 levels (shades of grey). (B) Fold increase in isolated, or …
(A) (Left) The N1WTD1+Rfng cell line (schematic). CHO-K1 cells were engineered to express wild-type Notch1 receptor (‘N1WT’, green), an H2B-Citrine reporter (yellow) activated by cleaved NICD …
(A) Fold increase in mean Notch activation levels in sparsely plated Caco-2 cells, transfected with the 12xCSL-H2B-Citrine reporter construct, with or without 10 uM DAPT. < 24 hr after plating, …
(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 …
(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 …
(A) Mean Notch activation levels, relative to background reporter fluorescence, in polyclonal N2D1 Pop cells plated on surfaces coated with (black) or without (blue) 2.5 ug/ml recombinant human Dll1 …
(A) E14.5 mouse cortical neural stem cells (NSCs) were plated sparsely and treated with ± 10 μM DAPT, cultured under growth conditions with low growth factors, and subsequently assayed for …
(A) Expression levels of Notch receptors, ligands, and Fringes, measured using RNAseq (see Materials and methods), in neural stem cells cultured in the presence of 0.5 ng/ml EGF and 10 μM DAPT for …
(A) Schematics showing how soluble recombinant N1ECD-Fc protein (rN1ECD-Fc) can be used to test whether surface interactions between ligand (red) and receptor (green) are necessary for cis-activation…
(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, …
(A) In each cis-activaiton model, Notch (‘N’, green) and Delta (‘D’, blue) interact to produce one or more cis-complexes, which can be active (‘C+’), producing NICD (green arrow) or inhibited (‘C-’, …
MATLAB code and parameter sets used for models in Figure 5.
For clarity, each plot only shows 3000 parameter pairs, randomly subsampled from the 10,000 total parameters sets analyzed. Orange dots are subsampled from parameters that produce non-monotonic C+ …
Examples of isolated CHO-K1 N1D1 + Rfng cells activating prior to cell division in the cis-activation assay. (Top row) Blue channel shows fluorescence of the constitutively expressed nuclear …
Examples of isolated NMuMG N1D1 + Rfng cells activating prior to cell division in the cis-activation assay. (Top row) Blue channel shows fluorescence of the constitutively expressed nuclear …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Mus musculus) | Dll1 | NCBI ID: 13388 | ||
Gene (Homo sapiens) | Dll1 | NCBI ID: 28514 | ||
Gene (Homo sapiens) | Dll4 | NCBI ID: 54567 | ||
Gene (Mus musculus) | R-fringe | NCBI ID: 19719 | ||
Gene (Homo sapiens) | Notch1 | NCBI ID: 4851 | ||
Gene (Homo sapiens) | Notch2 | NCBI ID: 4853 | ||
Cell line (Cricetulus griseus) | CHO-K1 | Thermo Fisher Scientific (T-REx CHO-K1) | Cat# R71807 RRID:CVCL_D586 | Figures 1, 2 and 4; Figure 1—figure supplements 1, 2 and 3; Figure 2—figure supplements 1 and 2; Figure 4—figure supplement 1 |
Cell line (Cricetulus griseus) | N1D1 | Derived from CHO-K1 | CHO-K1 expressing pEF-hNECD-Gal4esn + pcDNA5- TO-Dll1-T2A-H2B-mCherry + pEV-UAS-H2B-Citrine | Figure 2 |
Cell line (Cricetulus griseus) | N1D1 + Rfng | Derived from N1D1 | CHO-K1 expressing pEF-hNECD-Gal4esn + pcDNA5- TO-Dll1-T2A-H2B-mCherry + pEV-UAS-H2B-Citrine + pLenti- CMV-R-fringe-T2A-Puromycin | Figures 1, 2 and 4;Figure 1—figure supplements 1 and 2; Figure 4—figure supplement 1 |
Cell line (Cricetulus griseus) | N1WTD1 + Rfng | Derived from CHO-K1 | CHO-K1 expressing pcDNA3‐hN1‐mod1 + pcDNA5-TO-Dll1-mCherry + pEV-12xCSL-H2B-Citrine + piggyBac CMV-R-fringe+pCS- H2B-Cerulean | Figure 1—figure supplement 3 |
Cell line (Mus musculus) | NMuMG | ATCC | Cat# CRL-1636 (Wild-type cells used to transfect in piggyBac-12xCSL- H2B-Citrine) RRID:CVCL_0075 | Figure 1—figure supplement 4 |
Cell line (Mus musculus) | NMuMG + Dll1 | Derived from NMuMG | Base wild-type cell line expressing piggyBac-12xCSL-H2B-Citrine + piggyBac-TO-Dll1-T2A -H2B-mCherry-P2A-Hygromycin | Figure 1—figure supplement 4 |
Cell line (Mus musculus) | NMuMG N1D1 + Rfng | Derived from NMuMG | NMuMG ΔN2ΔJ1 expressing piggyBac- CMV-hNECD-Gal4-ANK -T2A-H2B-Cerulean + piggyBac CMV-TO Dll1-T2A-H2B-mCherry- P2A-Hygromycin + pEV -2xHS4-UAS-H2B-Citrine- T2A-tTS-2xHS4-Blast-T2A- rTetR-HDAC4-P2A-R-fringe | Figure 1—figure supplement 3 |
Cell line (Homo sapiens) | Caco-2 | ATCC (Caco-2 C2BBe1) | Cat# CRL-2102 (Wild-type cells used to transfect in pEV-12xCSL-H2B-Citrine) RRID:CVCL_1096 | Figure 1—figure supplement 4 |
Cell line (Cricetulus griseus) | N1D1 Pop | Derived from CHO-K1 | CHO-K1 with pEV-UAS- H2B-Citrine + pCS-H2B- Cerulean + piggyBac-TO-Dll1-T2A-H2 B-mCherry + piggyBac- CMV-hN1ECD-Gal4 - Cell population (Pop) | Figure 2 |
Cell line (Cricetulus griseus) | N1D4 Pop | Derived from CHO-K1 | CHO-K1 with pEV-UAS- H2B-Citrine + pCS-H2B- Cerulean + piggyBac- TO-Dll4-T2A-H2B-mCherry + piggyBac-CMV-hN1ECD-Gal4 - Cell population (Pop) | Figure 2; Figure 2—figure supplement 1 |
Cell line (Cricetulus griseus) | N2D1 Pop | Derived from CHO-K1 | CHO-K1 with pEV-UAS-H2B- Citrine + pCS-H2B-Cerulean + piggyBac-TO-Dll1-T2A-H2B -mCherry + piggyBac- CMV-hN2ECD-Gal4 - Cell population (Pop) | Figure 2; Figure 2—figure supplement 2 |
Cell line (Cricetulus griseus) | N2D4 Pop | Derived from CHO-K1 | CHO-K1 with pEV-UAS- H2B-Citrine + pCS-H2B- Cerulean + piggyBac -TO-Dll4-T2A-H2B-mCherry + piggyBac-CMV-hN2ECD-Gal4 - Cell population (Pop) | Figure 2; Figure 2—figure supplement 2 |
Cell line (Mus musculus) | NSC | EMD Millipore | Cat# SCR029 (E14.5 mouse neural cortical stem cells - NSC) | Figure 3; Figure 3—figure supplements 1 and 2 |
Transfected construct (recombinant DNA) | pEV‐UAS‐H2B‐ Citrine | Sprinzak et al., 2010 | N/A | Reporter for Notch1ECD-Gal4 receptor in CHO cells |
Transfected construct (recombinant DNA) | pEV-2xHS4-UAS- H2B-Citrine-T2A- tTS-2xHS4-Blast- T2A-rTetR-HDAC4- P2A-R-fringe | This paper | N/A | Reporter for Notch1ECD- Gal4-ANK receptor in NMuMG cells (tTS was not relevant for this work and was inactivated by 4-epiTc); rTetR-HDAC4 was used to decrease Delta expression in the presence of Dox; Constitutive R-fringe expression in NMuMG cells |
Transfected construct (recombinant DNA) | pEV-12xCSL‐ H2B‐Citrine | Sprinzak et al., 2010 | N/A | Reporter for Notch1 wild-type receptor in CHO and Caco-2 cells |
Transfected construct (recombinant DNA) | pEF-hN1ECD -Gal4 | This paper | N/A | Notch1ECD-Gal4 synthetic receptor used in CHO clones |
Transfected construct (recombinant DNA) | pX330 (CRISPR-Cas9 plasmid system) | Cong et al., 2013 | N/A | Plasmid used to insert RNA guide sequence for CRISPR knockdown |
Transfected construct (recombinant DNA) | piggyBac-12x CSL-H2B-Citrine | This paper | N/A | NotchWT reporter placed into NMuMG WT cells but not used in this study |
Transfected construct (recombinant DNA) | piggyBac-CMV -hN1ECD-Gal4 | This paper | N/A | Notch1ECD-Gal4 synthetic receptor used in CHO populations |
Transfected construct (recombinant DNA) | piggyBac-CMV -hN2ECD-Gal4 | This paper | N/A | Notch2ECD-Gal4 synthetic receptor used in CHO populations |
Transfected construct (recombinant DNA) | pcDNA3‐hN1‐mod1 | Sprinzak et al., 2010 | N/A | Wild-type Notch1 receptor used in CHO clones |
Transfected construct (recombinant DNA) | piggyBac-CMV- hNECD-Gal4-ANK -T2A-H2B-Cerulean | This paper | N/A | Notch1ECD-Gal4-ANK synthetic receptor used in NMuMG clones |
Transfected construct (recombinant DNA) | pcDNA5-TO-Dll1 -T2A-H2B-mCherry | Nandagopal et al., 2018 | N/A | Inducible Delta-like1 ligand used in CHO clones and populations |
Transfected construct (recombinant DNA) | pcDNA5-TO‐ Dll1‐ mCherry | Sprinzak et al., 2010 | N/A | Inducible Delta-like1-mCherry fusion used in CHO clones with Notch1WT receptor |
Transfected construct (recombinant DNA) | piggyBac-CMV-TO Dll1-T2A-H2B- mCherry-P2A- Hygromycin | Nandagopal et al., 2018 | N/A | Inducible Delta-like1 ligand used in CHO populations and NMuMG clones |
Transfected construct (recombinant DNA) | piggyBac-CMV-TO- Dll4-T2A-H2B-mCherry-P2A-Hygromycin | Nandagopal et al., 2018 | N/A | Inducible Delta-like4 ligand used in CHO populations |
Transfected construct (recombinant DNA) | pLenti-CMV-R- fringe-T2A- Puromycin | This paper | N/A | Constitutive R-fringe expression in CHO N1D1 + Rfng cells |
Transfected construct (recombinant DNA) | pCS‐H2B‐Cerulean | Sprinzak et al., 2010 | N/A | Segmentation color used in CHO cells |
Transfected construct (siRNA) | Allstar Negative Control | Qiagen | SI03650318 | Control siRNA, Figure 3C |
Transfected construct (siRNA) | Dll1 siRNA | Thermo Fisher Scientific | Cat# 4390771 (ID: s65000) | Dll1 siRNA, Figure 3C |
Antibody | rabbit anti- mouse Notch2 | Cell Signaling Technologies | Cat# 5732 RRID:AB_10693319 | WB (1:1000) |
Antibody | rabbit anti- mouse Jagged1 | Cell Signaling Technologies | Cat# 2620 RRID:AB_659968 | WB (1:1000) |
Antibody | rabbit anti- mouse GAPDH | Cell Signaling Technologies | Cat# 2118 RRID:AB_561053 | WB (1:3000) |
Antibody | rabbit anti- mouse Dll1-ICD | Kindly provided by Gerry Weinmaster, UCLA | Antibody 88 c | WB (1:2000) |
Antibody | ECL Rabbit IgG HRP-linked whole antibody from Donkey Secondary | GE Healthcare Life Sciences | Cat #NA934 RRID:AB_772206 | WB (1:2000) |
Antibody | Anti-mouse AlexFluor 488 Secondary | Thermo Fisher Scientific | Cat# A21202 RRID:AB_141607 | ICC (1:1000) |
Other | SuperSignal West Pico Chemiluminescent Substrate | Thermo Fisher Scientific | Cat# 34580 | as recommended per the manufacturer |
Other | SuperSignal West Femto Chemiluminescent Substrate | Thermo Fisher Scientific | Cat# 34095 | as recommended per the manufacturer |
Recombinant DNA reagent | Lipofectamine LTX plasmid transfection reagent | Thermo Fisher Scientific | Cat# 15338–100 | as recommended per the manufacturer |
Recombinant DNA reagent | ViraPower Lentiviral Expression System | Thermo Fisher Scientific | Cat# K497500 | as recommended per the manufacturer |
Peptide, recombinant protein | Recombinant mouse IgG2A Fc Protein | R and D Systems | Cat# 4460 MG-100 | 10 ug/ml |
Peptide, recombinant protein | Recombinant mouse Dll1 Fc chimera | R and D Systems | Cat# 5026 DL-050 | 10 ug/ml |
Peptide, recombinant protein | Recombinant mouse Notch-1 Fc chimera | R and D Systems | Cat# 5267-TK-050 | 10 ug/ml |
Peptide, recombinant protein | Recombinant human Dll1ext-Fc fusion proteins | Sprinzak et al., 2010 | Kindly provided by Irwin Bernstein, MD at Fred Hutchinson Cancer Research Center | 2.5 ug/ml |
Chemical compound, drug | DAPT | Sigma Aldrich | Cat# D5942 | 1 uM (CHO cells); 10 uM (all other cells) |
Chemical compound, drug | 4-epi tetracycline Hydrochloride | Sigma Aldrich | Cat# 37918 | 0–200 ng/ml |
Chemical compound, drug | Doxycycline | Takara Bio USA Inc | Cat# 631311 | 1 ug/ml or 10 ug/ml |
Chemical compound, drug | Dexamethasone | Sigma Aldrich | Cat# D4902 | 100 ng/ml |
Commercial assay or kit | Miniprep kit | Qiagen | Cat# 27106 | |
Commercial assay or kit | QIAquick PCR Purification kit | Qiagen | Cat# 28104 | |
Commercial assay or kit | RNeasy mini kit for RNA extraction | Qiagen | Cat# 74106 | |
Commercial assay or kit | iScript cDNA synthesis kit | Bio-Rad | Cat# 1708890 | |
Commercial assay or kit | iQ SYBR Green Supermix | Bio-Rad | Cat# 1708880 | |
Commercial assay or kit | SsoAdvanced Universal Probes Supermix | Bio-Rad | Cat# 172–5282 | |
Commercial assay or kit | DNA HCR kit | Molecular Instruments | ||
Software, algorithm | Cell segmentation and tracking | Nandagopal et al., 2018 | N/A | Figure 1, Figure 1—figure supplement 2 |
Software, algorithm | FISH Transcript detection and quantification | This paper | https://github.com/nnandago/elife2018-dot_detection; Nandagopal, 2018a | MATLAB code for visualizing, segmenting, and detecting transcript dots in FISH-labeled cells. Used to generate Figure 1—figure supplement 2, Figure 3—figure supplement 1 |
Software, algorithm | cis-activation model | This paper | https://github.com/nnandago/elife2018-cis_activation_modeling; Nandagopal, 2018b | MATLAB code for modeling steady state concentrations of ligand, receptors and complexes for a range of parameters. Used to generate Figure 5, Figure 5—figure supplement 5– |