Journal: eLife

Article Title: Cis- activation in the Notch signaling pathway

doi: 10.7554/eLife.37880

Figure Lengend Snippet: ( 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 ext -IgG fusion protein (Materials and methods). ( B ) Mean Notch activation levels, relative to background reporter fluorescence, in polyclonal N2D4 Pop cells plated on surfaces coated with (black) or without (red) 2.5 ug/ml recombinant human Dll1 ext -IgG fusion protein. Values represent the mean of three replicate experiments and error bars represent s.e.m. In A and B, cells were induced to express a range of Dll1/4 levels (measured using co-translated mCherry fluorescence) and cultured under cis -activation assay conditions (5 x 10 3 N2D1/4 + 150 x 10 3 CHO-K1 cells). Note similar activation levels on Dll1 ext -IgG-coated surfaces for all cis Dll1/4 expression levels, suggesting that the Notch2 receptor is not inhibited by co-expressed ligand. Also note that the strength of cis -activation is similar to trans -activation by an excess of plate-bound ligand, suggesting that cis ligands can maximally activate Notch2-expressing cells.

Article Snippet: Antibody , rabbit anti- mouse Notch2 , Cell Signaling Technologies , Cat# 5732 RRID: AB_10693319 , WB (1:1000).

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

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: Antibody , rabbit anti- mouse Notch2 , Cell Signaling Technologies , Cat# 5732 RRID: AB_10693319 , WB (1:1000).

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 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: Antibody , rabbit anti- mouse Notch2 , Cell Signaling Technologies , Cat# 5732 RRID: AB_10693319 , WB (1:1000).

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: Antibody , rabbit anti- mouse Notch2 , Cell Signaling Technologies , Cat# 5732 RRID: AB_10693319 , WB (1:1000).

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: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Engaging CD40 on Mtb-infected DCs increases NOTCH2 expression on CD4 T cells in the lung BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry. (A) Frequency of NOTCH receptor-positive CD4 T cells. (B) Boolean analysis of NOTCH receptor-expressing and cytokine-positive CD4 T cells. All populations are singlets/live cells/CD3 + /CD4 + . Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001.

Article Snippet: To stain lung T cells, the following intracellular stain antibodies were used: PE-CF594 Rat anti-mouse IL-17A (clone: TC11-18H10, BD), PE-Cy7 Rat anti-mouse TNF (clone: MP6-XT22, BD), BV786 Rat anti-mouse CD4 (clone: RM4-5, BD), APC anti-mouse IFN-γ (clone: XMG1.2, eBioscience), BV421 Rat anti-mouse IL-2 (clone: JES6-5H4, BD), PE Rat anti-mouse NOTCH1 (clone: 22E5.5, BD), PerCP-Cy5.5 anti-mouse IL-22 (clone: Poly5164, Biolegend), PE-Cy7 anti-mouse CD40L (clone: SA047C3, Biolegend), V450 Hamster anti-mouse CD3ε (clone: 500A2, BD), BV605 Rat anti-mouse NOTCH2 (clone: 16F11, BD), Alexa700 Rat anti-mouse CD4 (clone: RM4-5, BD).

Techniques: Infection, Expressing, Blocking Assay, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Th 17 responses correlate with NOTCH2 expression and lower lung CFU BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry and lung homogenates were plated to enumerate Mtb burdens. (A) Correlogram using R package “corrplot” showing correlation between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells. (B) Correlations in R using “ggscatter” between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells. (C) Mtb lung colony-forming unit (CFU) and correlations between CFU and frequencies of marker-positive or cytokine-positive CD4 T cells using “ggscatter” in R. All correlations presented are Pearson’s correlations. Data in C) are presented as mean ± SD and were analyzed using an unpaired Student’s t test. Data are representative of 2 independent experiments. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001. See also Figure S10 .

Article Snippet: To stain lung T cells, the following intracellular stain antibodies were used: PE-CF594 Rat anti-mouse IL-17A (clone: TC11-18H10, BD), PE-Cy7 Rat anti-mouse TNF (clone: MP6-XT22, BD), BV786 Rat anti-mouse CD4 (clone: RM4-5, BD), APC anti-mouse IFN-γ (clone: XMG1.2, eBioscience), BV421 Rat anti-mouse IL-2 (clone: JES6-5H4, BD), PE Rat anti-mouse NOTCH1 (clone: 22E5.5, BD), PerCP-Cy5.5 anti-mouse IL-22 (clone: Poly5164, Biolegend), PE-Cy7 anti-mouse CD40L (clone: SA047C3, Biolegend), V450 Hamster anti-mouse CD3ε (clone: 500A2, BD), BV605 Rat anti-mouse NOTCH2 (clone: 16F11, BD), Alexa700 Rat anti-mouse CD4 (clone: RM4-5, BD).

Techniques: Expressing, Infection, Blocking Assay, Ex Vivo, Flow Cytometry, Marker

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet:

Article Snippet: To stain lung T cells, the following intracellular stain antibodies were used: PE-CF594 Rat anti-mouse IL-17A (clone: TC11-18H10, BD), PE-Cy7 Rat anti-mouse TNF (clone: MP6-XT22, BD), BV786 Rat anti-mouse CD4 (clone: RM4-5, BD), APC anti-mouse IFN-γ (clone: XMG1.2, eBioscience), BV421 Rat anti-mouse IL-2 (clone: JES6-5H4, BD), PE Rat anti-mouse NOTCH1 (clone: 22E5.5, BD), PerCP-Cy5.5 anti-mouse IL-22 (clone: Poly5164, Biolegend), PE-Cy7 anti-mouse CD40L (clone: SA047C3, Biolegend), V450 Hamster anti-mouse CD3ε (clone: 500A2, BD), BV605 Rat anti-mouse NOTCH2 (clone: 16F11, BD), Alexa700 Rat anti-mouse CD4 (clone: RM4-5, BD).

Techniques: Blocking Assay, Purification, Mutagenesis, Recombinant, Lysis, Staining, Sequencing, Electron Microscopy, Negative Control, SYBR Green Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Software