Journal: bioRxiv

Article Title: Three-axis classification of mouse lung mesenchymal cells reveals two populations of myofibroblasts

doi: 10.1101/2021.08.03.454930

Figure Lengend Snippet: ( A ) Immunostaining images showing that Myh11-CreER labels alveolar myofibroblasts (PDGFRA-high) and pericytes (PDGFRB+), but not distal interstitial cells (MEOX2+) in neonatal (P8) lungs. Pericytes remain labeled after tracing and distal interstitial cells remain unlabeled in mature (P30) lungs. Tam, 300 ug tamoxifen. Scale: 10 um. ( B ) Immunostaining images showing that Pdgfra GFP:CreER lineage-traced cells are MEOX2+. Tam, 300 ug tamoxifen. Scale: 10 um. ( C ) Immunostaining images showing that Pdgfrb CreER lineage-traced cells are pericytes (NOTCH3+). Tam, 250 ug tamoxifen. Scale: 10 um. ( D ) Immunostaining images showing that Myh11-CreER and Pdgfra GFP:CreER labeled cells are positive for cleaved Caspase 3 (CASP3; open arrowhead). Tam, 250 ug tamoxifen. Scale: 10 um. ( E ) Diagram showing labeled cells by the three drivers (symbols) in neonatal and mature lungs, consistent with developmental apoptosis of alveolar myofibroblasts. Dashed circle, inefficient labeling of distal interstitial cells, compared to alveolar myofibroblasts, by Pdgfra GFP:CreER in neonatal lungs. Created with BioRender.com. ( F ) Table summarizing the cell types of the three axes and their markers.

Article Snippet: The following antibodies were used for immunofluorescence: goat anti-platelet derived growth factor-alpha (PDGFRA, 1:1000, AF1062, R&D Systems), rat anti-PDGFRA (PDGFRA, 1:1000, 14-1401-82, eBioscience), rat anti-cadherin-4 (CDH4, 1:20, MRCD5, Developmental Studies Hybridoma Bank), rat anti-platelet derived growth factor receptor-beta (PDGFRB, 1:1000, 14-1402-82, eBioscience), goat anti-platelet derived growth factor receptor-beta (PDGFRB, 1:1000, AF1042, R&D Systems), goat anti-notch3 (NOTCH3, 1:500, AF1308, R&D Systems), rabbit anti-mesenchyme homeobox 2 (MEOX2, 1:1000, NBP2-30647, Novus Biological), rabbit anti-cleaved caspase-3 (CASP3, 1:500, 9661, Cell Signaling), goat anti-peptidase inhibitor 16 (PI16, 1:1000, AF4929, R&D Systems), rat anti-protein tyrosine phosphatase, receptor type, C (CD45, 1:2000, 14-0451-81, eBioscience), Alexa488-conjugated mouse anti-smooth muscle actin (ACTA2, 1:1000, Santa Cruz Biotechnology, sc-32251 AF488), Cy3-conjugated mouse anti-smooth muscle actin (ACTA2, 1:1000, Sigma, C6198), Alexa647-conjugated mouse anti-smooth muscle actin (ACTA2, 1:1000, Santa Cruz Biotechnology, sc-32251 AF647), rat anti-advanced glycosylation end product-specific receptor (RAGE, 1:1000, R&D Systems, MAB1179), rabbit anti-transgelin (TAGLN, also known as SM22, 1:1000, Abcam, ab14106), chicken anti-green fluorescent protein (GFP, 1:5000, Abcam, AB13970), goat anti-hedgehog-interacting protein (HHIP, 1:1000, R&D Systems, AF1568), guinea pig anti-lysosomal-associated membrane protein 3 (LAMP3, 1:500, Synaptic Systems, 391005), mouse anti-NK2 homeobox 1 (NKX2-1, 1:500, Leica Biosystems, TTF-1-L-CE), goat anti-interleukin 33 (IL33, 1:500, R&D Systems, AF3626), rabbit anti-calponin (CNN1, 1:1000, Abcam, ab46794), and goat anti-delta/notch-like EGF repeat containing (DNER, 1:1000, AF2264, R&D Systems).

Techniques: Immunostaining, Labeling

Journal: International Journal of Clinical and Experimental Pathology

Article Title: The clinicopathologic significance of Notch3 expression in prostate cancer

doi:

Figure Lengend Snippet: Examples of the expression patterns of Notch3 in cases of prostate cancer (A) No expression; (B) Weak expression; (C) Overexpression (immunohistochemical stain, ×200).

Article Snippet: A rabbit polyclonal anti-Notch3 antibody (1:80, clone sc-515825, Santa Cruz, Biotechnology, CA, USA) was incubated with the sections for 1 h at room temperature.

Techniques: Expressing, Over Expression, Immunohistochemical staining, Staining

Journal: International Journal of Clinical and Experimental Pathology

Article Title: The clinicopathologic significance of Notch3 expression in prostate cancer

doi:

Figure Lengend Snippet: Correlation between pathological factors and the expression of Notch3 in prostate cancer

Article Snippet: A rabbit polyclonal anti-Notch3 antibody (1:80, clone sc-515825, Santa Cruz, Biotechnology, CA, USA) was incubated with the sections for 1 h at room temperature.

Techniques: Expressing

Journal: Cell Systems

Article Title: Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling

doi: 10.1016/j.cels.2016.11.013

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Notch3 (clone M-134) , Santa Cruz , Cat#sc-5593; RRID: AB_2151246.

Techniques: Transduction, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software, Inhibition, Modification

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Modular structure of NOTCH3, SP indicates signal peptide, red shaded EGF-like modules indicate core ligand binding region, LNR indicates Lin12-Notch repeats, NRR is the negative regulatory region, S1, S2 and S3 are proteolytic cleavage sites, HD-N and HD-C are the heterodimer interface region. RAM and Ankyrin repeat region (Ank) are motifs involved in binding CSL transcription factors, PEST domain is involved in ubiquitin-dependent turnover of the ICD. Location of mutants used in this study are indicated by arrows. Coloured bars indicate locations of regions of NOTCH3 in which epitopes used in this study are located. (B-D) AlphaFold2 predictions of WT NOTCH3 EGF modules 2 (B) , 5 (C) and 11 (D) , and mutant substitutions introduced using the SDM programme. Location of the new Cys residue in R90C indicated in yellow, and other substitutions that replace as cysteine residues are shown in magenta. (E-G) Surface views of the structures from (B-D) depicted as a surface mesh. The introduced cysteine in R90C is surface exposed (E) , but the free cysteines in C212Y and C455R are buried within the module structure. ΔΔG values indicate predicted decreases in stability. (H-K) Expressed WT NOTCH3 (H) and CADASIL mutants (I-K) do not show strong accumulation in the ER when expressed in hTert-RPE1 cells. (L, M) scoring of NOTCH3 localisation in KDEL-positive ER, by % area of ER occupied (L) and by % of total NOTCH3 staining intensity (K) in z-sections through the cytoplasmic region. R90C shows a small increase in %NOTCH3 which is localised to the ER. * indicates P<0.05 by student t-test, error bars SEM, n=10. (N) Antibody surface labelling of NOTCH3 localisation on non-permeabilised cells at time 0 and 60 minutes during a live cell anti-ECD uptake assay. Similar time zero surface distributions, and subsequent surface depletion of WT and CADASIL mutants can be seen.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Ligand Binding Assay, Binding Assay, Mutagenesis, Residue, Staining

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A-C) Schematic view of pulse chase labelling protocol. (D-G) WT NOTCH3 localisation in permeabilised cells after antibody uptake at 0 (D) , 15 (E) , 30 (F) , and 60 (G) minutes. Little surface-labelled NOTCH3 is transported to the early endosome by 15 minutes chase but is evident in EEA1 positive endosomes by 30 and 60 minutes. Presence of both full-length and ECD-only staining suggests ECD-shedding can occur by the time NOTCH3 is localised to the EEA1-marked endosome. (H-J) CADASIL mutant NOTCH3 localisation after 60-minute chase shows the mutant proteins labelled at the cell surface also become localised to the EEA1-positive endosome. (K, L) After 60-minute chase surface, little labelled NOTCH3 ECD has reached the CD63-positive late endosome while ICD is present. However full-length NOTCH3 and ECD-only localisations are found adjacent to CD63-marked organelles.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Pulse Chase, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A-D) Immunolocalisation of NOTCH3 with anti-ECD and anti-ICD compared with EEA1-marked endosomes in fixed and permeabilised cells of WT (A) and CADASIL mutants ( B-D) . (E) Quantitation of NOTCH3 localisation in the early endosome, showing % endosomal-located NOTCH3 puncta which either full-length NOTCH3, ECD-only, or ICD-only staining. R90C is distinguished from WT and other mutants by a greater proportion of ICD-only puncta compared to ECD or full-length. For WT, n=91 NOTCH3 puncta from198 endosomes scored. For R90C, n=139 NOTCH3 puncta from 180 endosomes score. For C212Y, n=140 NOTCH3 puncta from 240 endosomes scored. For C455R, n=164 NOTCH3 puncta from 302 endosomes scored. Minimum of 7 cells scored for each construct. Data obtained from z sections from at least 7 cells. (F) Full-length NOTCH3, or ECD-only puncta located within or adjacent to Rab11-positive endosomes. (G, H) In CD63 positive endosomes (G) and LAMP1-positive lysosomes (H) , NOTCH3 localisation is predominantly as ICD-only puncta, quantified in (I, J) . Error bars in I and J are SEM, n=11 and n=7 respectively. Statistical significance by student t-test.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Quantitation Assay, Staining, Construct

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Schematic diagram illustrating alternative explanations for visualisation of separated ECD and ICD-positive puncta, including: ECD shedding by S2 cleavage; epitope masking; fragmentation to remove terminal epitopes; non-specific staining. (B) Colocalisation of two different ECD epitopes compared to ICD ab23426 . ICD-stained puncta can be observed which lack both ECD epitopes. (C) Colocalisation of two different ICD epitopes compared to anti-ECD 1E . ECD-only spots are present which lack both ICD epitopes. (D, E) Quantification of the colocalisation between the epitopes used in B, C. In D * indicates P<0.05 compared with double ECD epitope colocalisation by student t-test. Error bars represent SEM, n=5 (D) , n=14 (E) . (F-H) Both ICD epitopes are located to the nucleus in NOTCH3 transfected cells but not the ECD epitope.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) EEA1-positive early endosomes of MCF7 cells costained with anti-ECD, and ICD. Arrowheads indicate endosomes shown enlarged in insets which have separate localisation of ECD and ICD puncta. (B) CD63-positive late endosomes costained with anti-ECD, and ICD. Boxed region is enlarged in inset showing late endosomes predominantly contain ICD-only puncta. (C) After treatment of MCF7 cells with metalloprotease inhibitor BB94, more full-length NOTCH3 staining is observed in the late endosomes, colocalised with CD63. (D) Treatment of cells with gamma-secretase inhibitor DAPT does not alter localisation of ECD in the late endosome. (E-G) Quantification of colocalization of ECD vs ICD (E) , ECD vs CD63 (F) , and ICD vs CD63 ( G) . Error bars, SEM, control DMSO-only treated cells n=10, BB94 n=18, DAPT n=12. * indicates p<0.05 by student t-test compared to control.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Schematic figure illustrating luciferase reporter assay methodology. (B) WT and CADASIL mutant NOTCH3 signalling after transfection into hTERT-RPE1 cells compared to empty vector (EV) control. * indicates p<0.05 compared to WT NOTCH3 by student t-test. Error bars SEM, minimum of n=4. (C-F) NOTCH3 signalling after treatment of transfected cells with TRPML inhibitor (ML-Sl1), gamma-secretase inhibitor (RO 4929097 ), or metalloprotease inhibitor (BB94) for WT (C) , R90C (D) , C212Y (E) , and C455R (F) * indicates p<0.05 compared with control by student t-test. Error bars are SEM, minimum of n=3.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Luciferase, Reporter Assay, Mutagenesis, Transfection, Plasmid Preparation, Control