Journal: Journal of Neuroinflammation

Article Title: Astrocytic DLL4-NOTCH1 signaling pathway promotes neuroinflammation via the IL-6-STAT3 axis

doi: 10.1186/s12974-024-03246-w

Figure Lengend Snippet: DLL4-NOTCH1 signaling in reactive astrocytes promotes IL-6 transcription via a direct interaction with NICD. ( A ) Notch1 ACKO C mice and control mice induced with EAE were scored daily according to a widely-used 5-point scale (EAE scoring: 1 limp tail; 2 limp tail and weakness of hind limb; 3 limp tail and complete paralysis of hind legs; 4 limp tail, complete hind leg and partial front leg paralysis). Statistical significance was determined by using a 2 ways Anova test followed by the Holm-Sidak’s multiple comparisons test, (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 ****: p ≤ 0.0001). ( Notch1 ACKO C n = 15, WT n = 16). ( B ) NA were cultured until confluence and treated with IL-1β 10ng/mL for 24 h. A Chromatin Immuno-Precipitation (ChiP) was then performed on NA lysates using NICD antibody versus IgG controls to pull-down. IL-6 DNA expression level was then quantified by PCR. ( C-G ) NA were cultured until 70% confluence. They were then transduced with an empty lentivirus (6.21 10 8 PFU/mL) versus a DLL4 -expressing lentivirus (4.14 10 8 PFU/mL) and harvested 24 h post transduction ( n = 11). ( C ) HES1 , ( D ) HEY1 , ( E ) HEY2 , (F ) IL-6 and ( G ) DLL4 expression were quantified by qRT-PCR. β-ACTIN was used as a reference. Statistical significance was determined by using a Mann-Whitney U test

Article Snippet: Anti ACTB (rabbit), anti-CASP3 (rabbit) and Cleaved CASP3 (rabbit), anti-cleaved NOTCH1 (NICD) (rabbit), anti-DLL4 (rabbit), anti-human-JAG1 (rabbit) and anti-VIM (rabbit) were from cell signaling (Danvers, MA, USA).

Techniques: Control, Cell Culture, Chromatin Immunoprecipitation, Expressing, Transduction, Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Astrocytic DLL4-NOTCH1 signaling pathway promotes neuroinflammation via the IL-6-STAT3 axis

doi: 10.1186/s12974-024-03246-w

Figure Lengend Snippet: Summary scheme of the role of DLL4-NOTCH1 signaling on astrocyte reactivity during neuroinflammation

Article Snippet: Anti ACTB (rabbit), anti-CASP3 (rabbit) and Cleaved CASP3 (rabbit), anti-cleaved NOTCH1 (NICD) (rabbit), anti-DLL4 (rabbit), anti-human-JAG1 (rabbit) and anti-VIM (rabbit) were from cell signaling (Danvers, MA, USA).

Techniques:

Journal: The Journal of Cell Biology

Article Title: SNAP23 deficiency causes severe brain dysplasia through the loss of radial glial cell polarity

doi: 10.1083/jcb.201910080

Figure Lengend Snippet: SNAP23 deficiency decreases β-catenin and Notch signaling. (A and B) Immunoblots showing the levels of total and active β-catenin in the control (Ctrl) and NcKO brains at E13.5. Lamin B blotting was used as a loading control. (C and D) Immunoblots showing the full-length (FL) and cleaved Notch1 (NICD) levels in the Ctrl and NcKO brains at E13.5. Lamin B blotting was used as a loading control. (E and F) Relative expression of Axin2 and Hes5 mRNA in the Ctrl and NcKO brains at E13.5, as measured with real-time PCR ( n = 5 mice per genotype). (G) d1EGFP expression in the cerebral cortex with or without NICD plasmid. Scale bar, 200 µm. (H) TUNEL stained in the electroporated region of the cerebral cortex. pX330 was electroporated as a control. The mCherry plasmid was electroporated to show the electroporated region. The blue arrowheads indicate TUNEL-positive cells. Scale bar, 50 µm. (I) Density of TUNEL-positive cells in the electroporated region of the cerebral cortex ( n = 5 sections from three mice per genotype).

Article Snippet: The following primary and secondary antibodies were used: anti-SNAP23 (rabbit; ), anti-SNAP25 (mouse; Covance, SMI-81R), anti-Nestin (mouse; BD Pharmingen, 556309), anti-Pax6 (rabbit; COVANCE, PRB-278P), anti-Pericentrin (rabbit; Covance, PRB-432C), anti-PH3 (rabbit; Upstate, 06–570), anti-Tuj1 (mouse; Covance, MMS-435P), anti-Tbr1 (rabbit; Millipore, AB10554), anti-Calbindin (goat; Santa Cruz Biotechnology, sc-7691), anti-Ki67 (rabbit; Novo Castra, NCL-Ki67p), anti-BrdU (mouse; BD Pharmingen, 555627), anti–cleaved caspase-3 (rabbit; Cell Signaling Technology, 9661), anti–N-cadherin (mouse; BD Transduction, 610920), anti–N-cadherin ectodomain (mouse; Sigma-Aldrich, C3865), anti–E-cadherin (mouse; BD Transduction, 610181), anti–total β-catenin (mouse; BD Transduction, 610153), anti–active β-catenin (mouse; Upstate, 05–665), anti-Par3 (rabbit; Upstate, 07–330), anti–ZO-1 (mouse; Zymed, 33–9100), anti-Crb3 (rabbit; a kind gift from Dr. Dominique Massey-Harroche, Aix-Marseille University, Marseille, France), anti-Pals1 (mouse; Santa Cruz Biotechnology, sc-365411), anti–β1-integrin (rat; Chemicon, MAB1997), anti–ephrin-B1 (goat; R&D Systems, AF473), anti-LDLR (goat; R&D Systems, AF2255), anti–Na + /K + -ATPase β subunit (rabbit antisera; a kind gift from Dr. Haruo Homareda, Kyorin University, Mitaka, Tokyo, Japan), anti-mCherry (rat; Invitrogen, M11217), anti-Notch1 (rabbit; Cell Signaling Technology, 3608), anti–cleaved Notch1 (rabbit; Cell Signaling Technology, 2421), anti-VAMP3 (rabbit; Synaptic Systems, 104102), anti-VAMP4 (rabbit; Invitrogen, PA1-768), anti-VAMP5 (rabbit; Synaptic Systems, 176003), anti-VAMP8 (rabbit; Synaptic Systems, 104302), anti-Syntaxin1A (mouse; Sigma-Aldrich, S0664), anti-Syntaxin1B (rabbit; Synaptic Systems, 110402), anti-Lamp2 (mouse; Developmental Studies Hybridoma Bank, H4B4), anti-GAPDH (mouse; Calbiochem, CB1001), anti–Lamin B (mouse; Santa Cruz Biotechnology, sc-374015), Alexa 488–, 568–, and Cy5-labeled donkey anti-rabbit, anti-mouse, and anti-rat IgG (Molecular Probes), and HRP-labeled donkey anti-rabbit, anti-mouse, anti-rat, and anti-goat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Western Blot, Control, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, TUNEL Assay, Staining

Journal: Developmental Cell

Article Title: Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model

doi: 10.1016/j.devcel.2020.06.039

Figure Lengend Snippet: Notch Glycosylation Impacts Epidermal Differentiation (A) Schematic model of the Notch pathway. POGLUT1 KO lacks O-Glc on EGF-like repeats, POFUT1 KO lacks O-Fuc on EGF-like repeats, JAG1 KO lacks the main ligand (Jagged-1) for Notch receptors, and NOTCH1 KO lacks the Notch1 receptor. Drawing adapted from Haltiwanger et al. (2015) . (B) Micrographs illustrating HE staining and immunolabeled tissue sections of organotypic cultures. The expression of differentiation markers K10 and INV is shown. Scale bar represents 50 μm (n = 3). (C) Immunohistochemical staining of tissue sections from N/TERT-1 WT and POFUT1 KO , POGLUT1 KO , NOTCH1 KO , and JAG1 KO tissues stained with antibodies recognizing cleaved (activated) Notch intracellular domains, p63, and the proliferation marker Ki67. Scale bar represents 50 μm (n = 3). (D) Diagrams showing the number of cell layers negative for K10 (left panel) or involucrin (IVL, right panel) and the total number of cell layers. The columns depict mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p = 0.0001 using one-way ANOVA followed by multiple comparison analysis for WT versus KO cells (n = 4). (E) Number of p63-positive cells for each cell layer in POFUT1 KO and POGLUT1 KO tissues (n = 4). (F) Differential expression of cellular senescence and differentiation genes, as evaluated by RNA-seq, of conventionally grown WT, POFUT KO , and POGLUT1 KO cells. FPKM values for a select list of genes involved in cellular senescence (left panel) or cellular differentiation (right panel) were loaded into Qlucore Omics Explorer. Genes that significantly contribute to sample variance ( q ≤ 0.05) are shown. Red indicates higher expression, and blue indicates lower expression. (G) Western blot of N/TERT-1 WT, POFUT1 KO , and POGLUT1 KO cells with an antibody against NICD (cleaved Notch 1). EGTA induces ligand-independent Notch1 activation and is used as a positive control, whereas γ-secretase inhibitor DAPT blocks Notch signaling. DMSO is used as a control. The downstream target of Notch1, HES1, is included (n = 2). (H) O-fucose and O-glucose glycosylation of Notch differentially affects keratinocyte differentiation. In the interfollicular epidermis, the reciprocal signaling between Notch and p63 contribute to the balance between self-renewing and proliferating keratinocytes at various stages of differentiation. Notch 1–4 activity suppresses the expression of p63 and vice versa, controlling the expression of HES1 , CDKN1A (p21), and WNT4 differentiation, counteracting the effects of Notch on their expression. Our data suggest that both Pofut1 and Poglut1 differentially regulate the dynamic equilibrium between putative stem cells and cells committed to terminal differentiation. Figure adapted from .

Article Snippet: rabbit anti-cleaved Notch1 (Val1744) (D3B8) (IF 1:100, WB 1:1000) , CST , Cat# 4147; RRID: AB_2153348.

Techniques: Glycoproteomics, Staining, Immunolabeling, Expressing, Immunohistochemical staining, Marker, Comparison, Quantitative Proteomics, RNA Sequencing, Cell Differentiation, Western Blot, Activation Assay, Positive Control, Control, Activity Assay

Journal: Developmental Cell

Article Title: Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model

doi: 10.1016/j.devcel.2020.06.039

Figure Lengend Snippet:

Article Snippet: rabbit anti-cleaved Notch1 (Val1744) (D3B8) (IF 1:100, WB 1:1000) , CST , Cat# 4147; RRID: AB_2153348.

Techniques: Plasmid Preparation, Virus, Recombinant, Protease Inhibitor, Cell Adhesion Assay, Fractionation, Staining, Bicinchoninic Acid Protein Assay, Western Blot, Expressing, Software, Microscopy, Transmission Assay, Mass Spectrometry