notch1 icd  (Cell Signaling Technology Inc)

Journal: Vascular Biology

Article Title: Under pressure: integrated endothelial cell response to hydrostatic and shear stresses

doi: 10.1530/VB-25-0015

Figure Lengend Snippet: Microfluidic culture of ECs to decouple pressure and flow effect. (A) Schematic of the flow circuit connecting the culture channel and a resistor channel in series with the syringe pump. Different resistor channels were designed with specific width and height to change the hydrostatic pressure P set at the outlet of culture channels. (B) Representative images of ECs cultured at shear stress τ w = 5 dyne/cm 2 show distinct cell morphology and alignment for P set = 0 (i) and 60 mmHg (ii). Red: F-actin; green: KI-67; blue: nuclei; gray: VECad. (C) Quantification of cell area, alignment angle, aspect ratio, and number of nuclei per 40x field for two pressure conditions. * P < 0.05. (D) Representative immunostaining images of ECs show different polarization of Notch1-ECD (green) at different pressure conditions under flow. (E, F, G, H) Transcriptional changes of ECs in response to flow and pressure. (E) Principal component analysis showing the separation of static and flow conditions in PC1 and mild pressure separation in PC2. (F) Venn diagram of differentially expressed gene numbers comparing the effect of flow and pressure on EC response. Red: upregulated genes; blue: downregulated genes. 5_0: τ w = 5 dyne/cm 2 and P set = 0 mmHg; and 5_60: τ w = 5 dyne/cm 2 and P set = 60 mmHg. (G) Heatmap of selected flow-responsive markers (upper) and differentially expressed cell-cycle-related genes comparing static and pressure groups. Colormap: log2(CPM) with minimum value in blue and maximum in red. (H) GO term analysis and the number of genes associated comparing high vs low pressure at the same flow shear condition.

Article Snippet: The primary antibodies used included: VECAD/CD144 (1:100, Abcam ab33168, UK), AQP-1 (1:100, Santa Cruz sc-25287, USA), GJA4 (1:100, Abcam ab181701), DLL4 (1:100, Novus NB600-892, USA), Ki-67 (1:100, Abcam ab16667), Notch1-ICD (1:100, Cell Signaling 3608S, USA).

Techniques: Cell Culture, Shear, Immunostaining

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation of keratinocytes and angiogenesis in diabetic mice. (a) Expression levels of integrin β1, K14, K10, K1, and Notch1 NICD evaluated by Western blot in the middle and at the end of wound healing. (b-f) Corresponding quantitative analysis. (g) CD31 and VEGF expression levels evaluated by Western blot. (h and i) Corresponding quantitative analysis. n = 3 in each group. ✶ P < 0.05. NICD: Notch Intracellular Domain, VEGF: Vascular endothelial growth factor, K14: Keratin 14, K10: Keratin 10, K1: Keratin 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation through interaction between HIF-1α and NICD. (a) Immunofluorescence showing the expression and co-localization of HIF-1α and NICD in HaCaT cells under different conditions; bar = 100 μm. (b) Western blot showing the expression levels of HIF-1α, NICD, K14, and integrin-β1 in HaCaT cells under different conditions. (c-f) Quantitative analysis of WB. n = 3 in each group; ✶ P < 0.05. (g) Co-IP confirming the interaction between HIF-1α and NICD. HIF-1: Hypoxia-inducible factor 1, NICD: Notch intracellular domain, WB: Western blot, Co-IP: Co-immunoprecipitation, K14: Keratin 14.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Immunofluorescence, Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Schematic model of roxadustat reversing the “high differentiated and low proliferation” state of keratinocytes in diabetic wounds. In normal skin, keratinocytes maintain a relatively stable rhythm of proliferation and differentiation. Injury can activate the process of wound repair, upregulate HIF-1 signal, downregulate Notch1 signal, and make keratinocytes in a state of “high proliferation and low differentiation.” In the context of diabetes, the skin is usually thin and wound healing is delayed, HIF-1 signaling is inhibited, and Notch1 signaling is continuously activated, making keratinocytes in a state of “low proliferation and high differentiation.” FG-4592 upregulates the inhibited HIF-1 signaling and downregulates the hyperactivated Notch1 signaling, which both benefit diabetic wound re-epithelialization. By Fig draw (version 2.0, www.figdraw.com ). HIF-1: Hypoxia-inducible factor 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Western blot of hdBEC lysates cultured under static or flow (∼20 dynes/cm ) conditions, with pre-treatment with 0.5 μg/mL of rhDll4 or vehicle control. (B) Quantification of normalized Western blot intensity of Notch1 V1754. (C) Western blot of hdBEC lysates from scramble (SCR), DLL4 KO , and JAG1 KO hdBECs under static and flow conditions. (D) Quantification of normalized Western blot intensity of Notch1 V1754. (E) Western blot of hdBEC and hdLEC lysates under static and flow conditions. (F) Quantification of normalized Western blot intensity of Notch1 V1754. (G) Fluorescence micrographs of hdBECs and hdLECs under static and flow conditions immunostained for Notch1 ICD (heatmap) and VE-cadherin (white). Scale bar, 25 μm. (H) Quantification of the relative Notch1 polarization in hdBECs versus hdLECs under flow. n ≥ 10 fields of view from three independent experiments. (I) Timelapse of Notch1-GFP in hdBEC cells under flow. Time scale (min:sec). Scale bar, 10 μm. (J) Fluorescence micrographs of SCR and DLL4 KO cells under flow conditions immunostained for Notch1 (black) and DNA (blue). Scale bar, 25 μm. (K) Quantification of the relative degree of Notch1 polarization in SCR versus DLL4 KO cells under flow. n ≥ 10 fields of view from three independent experiments. (K) Quantification of the relative degree of ligand polarization in Dll4-GFP or Jag1-mEmerald cells under flow. n ≥ 10 fields of view from three independent experiments. Western blots are representative of three independent experiments. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Western Blot, Cell Culture, Control, Fluorescence

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Left: Fluorescence micrographs of co-culture of hdBECs expressing mEmerald (green) or mApple (magenta) under flow. Scale bar, 5 μm. Inset: High magnification micrograph of the cell-cell interface with mApple, mEmerald, and Notch1 ECD (cyan). Yellow inset: XZ orthogonal projection. Scale bar, 5 μm. Right: experimental schematic. (B) Fluorescence micrographs of hdBECs pretreated for 1 h with 10 μM DAPT, 1 μM BB94 or DMSO vehicle control and cultured under flow. Scale bar, 25 μm. (C) Quantification of Notch1 polarization in hdBECs treated with DAPT, BB94, or DMSO vehicle control under flow. n ≥ 10 fields of view from three independent experiments. (D) Super-resolution by optical pixel reassignment fluorescence micrographs of the downstream cell-cell interface of flow-polarized hdBECs. Scale bar, 5 μm. (E) Line scan quantification of relative Notch1 ECD (magenta) and Notch1 ICD (green) distribution (representative yellow dashed line). n = 10 profiles. (F) Fluorescence time series from live cell movie of hdBECs labeled with AF647-Notch1 ECD antibody under flow. Pseudo-colored to depict cell-cell boundaries. Insets (i) and (ii) of distinct polarized domains where individual particles (red circles) are tracked over time moving retrograde opposite the direction of flow. Time scale (min:sec). Scale bar, 5 μm. (G) Schematic illustrating pulse-chase Notch1 ECD antibody labeling experiments. (H) Fluorescence micrographs of internalized Notch1 in SCR and DLL4 KO cells under flow conditions from pulse-chase labeling of Notch1 ECD (black). Scale bar, 25 μm. (I) Quantification of endocytosed Notch1 in SCR versus DLL4 KO cells. n ≥ 10 fields of view from three independent experiments. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Fluorescence, Co-Culture Assay, Expressing, Control, Cell Culture, Labeling, Pulse Chase, Antibody Labeling

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Wild-type Notch1 (WT) or a Notch1 mutant lacking the intracellular domain (ΔICD) C-terminally tagged with mEmerald. (B) Western blots of Notch1-WT expressing hdBEC lysates cultured under static and flow conditions or plated on control or rhDll4-coated substrate. Quantification: Notch1 V1754 intensity normalized to total construct expression (mEmerald recognized by GFP antibody). (C) Western blot of Notch1-ΔICD expressing hdBEC lysates cultured under static and flow conditions or plated on rhDll4-coated dishes. Quantification: Notch1 V1754 intensity normalized to total construct expression (mEmerald recognized by GFP antibody). Quantification of Western blot intensity of Notch1 V1754 normalized to GFP. n = 3 independent experiments. (D) Western blot of lysates from Notch1-ΔICD expressing hdBEC monolayers mosaically co-cultured with increasing percentage of Dll4-mScarlet overexpressing hdBECs. Quantification is Notch1 V1754 intensity normalized to GFP. (E) Fluorescence micrographs of Notch1-WT and Notch1-ΔICD expressing hdBECs under flow. Yellow arrows denote flow-polarized domains. Scale bar, 25 μm. (F) Quantification of Notch1 polarization in Notch1-WT and Notch1-ΔICD expressing hdBECs under flow. n ≥ 10 fields of view from three independent experiments. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, **p < 0.01, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Mutagenesis, Western Blot, Expressing, Cell Culture, Control, Construct, Fluorescence

Journal: bioRxiv

Article Title: The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

doi: 10.1101/2025.07.13.663563

Figure Lengend Snippet: (A) Select list of ICD-interacting proteins identified via mass spectrometry to increase under flow. (B) Western blot of Notch1 co-immunoprecipitation from hdBECs cultured under static or flow conditions. (C) Western blot of lysates from SCR and ANXA2 KO hdBECs under flow. (D) Quantification of Western blot intensity of Notch1 V1754 normalized to GAPDH. Data is normalized to SCR for n = 3 independent experiments. (E) Fluorescence micrograph of annexin A2 in hdBECs under flow. Scale bar, 25 μm. (F) Fluorescence micrographs of endocytosis of Notch1 from pulse-chase labelled of SCR and ANXA2 KO cells. Purple dashed lines indicate cell segmentation. Scale bar, 25 μm. (G) Quantification of internalized Notch1 ECD in SCR versus ANXA2 KO cells. n ≥ 10 fields of view from three independent experiments. (H) Associated quantification of the relative degree of Notch1 polarization in SCR versus ANXA2 KO cells under flow, measured as described previously. n ≥ 10 fields of view from three independent experiments. (I) Fluorescence micrographs of caveolin-1 and VE-cadherin in hdBECs under flow. Scale bar, 25 μm. (J) Western blot of hdBEC lysates from SCR and CAV1 KO cells cultured under static and flow conditions or plated on rhDll4-coated substrates. (K) Quantification of Western blot intensity of Notch1 V1754 normalized to GAPDH. Data is normalized to SCR static condition. (L) Quantification of internalized Notch1 ECD by pulse-chase labeling in SCR versus CAV1 KO cells. n ≥ 10 fields of view from three independent experiments. (M) Quantification of Notch1 polarization in SCR versus CAV1 KO cells under flow. n ≥ 10 fields of view from three independent experiments. (N) Western blot of detergent-resistant membranes (DRM) fractions isolated from SCR and CAV1 KO hdBEC cells under static and flow conditions. (O) Fluorescence micrographs of hdBECs and hdLECs under flow. Scale bar, 25 μm. (P) Schematic depicting flow-polarized downstream domains where (a) ICD localizes full-length Notch1, (b) annexin A2 regulates Notch1 cis-endocytosis, (c) and caveolin-1 establishes microdomains compartmentalizing Notch1 and γ-secretase. For all plots, mean ± SD; one-way Anova with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes non-significant.

Article Snippet: Antibodies against Notch1 V1754 (V1744 in mice, D3B8, 1:500 WB), Notch1 ICD (D1E11, 1:200 IF, 1:1000 WB), GAPDH (14C10, 1:5000 WB), Dll4 (D7N3H, 1:1000 WB), Jag1 (D4Y1R, 1:1000 WB), GFP (D5.1, 1:5000 WB), Annexin A2 (D11G2, 1:1000 WB, 1:200 IF), Presenilin-1 (E3L9X, 1:1000 WB), and Flotillin-2 (C42A3, 1:1000 WB) were from Cell Signaling Technologies.

Techniques: Mass Spectrometry, Western Blot, Immunoprecipitation, Cell Culture, Fluorescence, Pulse Chase, Labeling, Isolation