Journal: Respiratory physiology & neurobiology

Article Title: Paralemmin-3 augments lipopolysaccharide-induced acute lung injury with M1 macrophage polarization via the notch signaling pathway.

doi: 10.1016/j.resp.2023.104203

Figure Lengend Snippet: Fig. 5. Effect of PALM3 expression on Notch signaling pathway in AMs isolated from the BALF of each group. (A) Notch signaling associated molecules (NICD, RBP-J and IRAK2) mRNA and protein expression in in AMs isolated from the BALF of each group. (B) Co-immunoprecipitation assays of NICD and IRAK2 with RBP-J in AMs isolated from the BALF of each group. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. normal group, and #P < 0.05 vs. empty vector group.

Article Snippet: Immunoprecipitation was performed with anti-rat-NICD antibody (1:50, #4147 monoclonal rabbit anti-rat, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ratRBP-J antibody (1:100, sc-271128, Santa Cruz) or normal rat IgG (1:100, sc-2026, Santa Cruz).

Techniques: Expressing, Isolation, Immunoprecipitation, Plasmid Preparation

Journal: Respiratory physiology & neurobiology

Article Title: Paralemmin-3 augments lipopolysaccharide-induced acute lung injury with M1 macrophage polarization via the notch signaling pathway.

doi: 10.1016/j.resp.2023.104203

Figure Lengend Snippet: Fig. 6. Involvement of Notch signaling pathway in PALM3-mediated M1 polarization. (A) The frequency of F4/80+CD16+ cells in AMs isolated from BALF were determined by flow cytometry with PE and FITC staining. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. lenti.r-PALM3 + DMSO group, and #P < 0.05 vs. NS + DMSO group. (B) The mRNA levels of M1 specific genes (TNF-α, IL-1, CCr7 and iNOS) in AMs isolated from BALF were determined by qRT-PCR. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. NS + DMSO group, and #P < 0.05 vs. lenti.r-PALM3 + DMSO group. (C) The mRNA levels of Notch signaling associated molecules (NICD, RBP-J and IRAK2) in AMs isolated from BALF were determined by qRT-PCR. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. NS + DMSO group, and #P < 0.05 vs. lenti.r-PALM3 + DMSO group. (D) Co-immunoprecipitation assays of NICD and IRAK2 with RBP-J in AMs isolated from BALF. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. NS + DMSO group, and #P < 0.05 vs. lenti.r-PALM3 + DMSO group.

Article Snippet: Immunoprecipitation was performed with anti-rat-NICD antibody (1:50, #4147 monoclonal rabbit anti-rat, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ratRBP-J antibody (1:100, sc-271128, Santa Cruz) or normal rat IgG (1:100, sc-2026, Santa Cruz).

Techniques: Isolation, Flow Cytometry, Staining, Quantitative RT-PCR, Immunoprecipitation

Journal: Respiratory physiology & neurobiology

Article Title: Paralemmin-3 augments lipopolysaccharide-induced acute lung injury with M1 macrophage polarization via the notch signaling pathway.

doi: 10.1016/j.resp.2023.104203

Figure Lengend Snippet: Fig. 7. Co-immunoprecipitation assays of NICD and RBP-J with PALM3 in AMs isolated from BALF. After rats were pretreated with lentiviral vectors, the AMs were collected from BALF on day 7 after successive LPS inhalations. The total cell lysates were immunoprecipitated with anti-NICD, anti-RBP-J or normal IgG antibodies. Then, the immunocomplexes were examined by western blot analysis using the indicated antibodies. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. normal rat group.

Article Snippet: Immunoprecipitation was performed with anti-rat-NICD antibody (1:50, #4147 monoclonal rabbit anti-rat, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ratRBP-J antibody (1:100, sc-271128, Santa Cruz) or normal rat IgG (1:100, sc-2026, Santa Cruz).

Techniques: Immunoprecipitation, Isolation, Western Blot

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: (A) Diagram of Notch protein structure. N1IC encodes the cytoplasmic domain of Notch1. N4/int-3 encodes 30 amino acids upstream of the transmembrane domain, the transmembrane domain, and the cytoplasmic domain of Notch4 fused to a HA tag. (B) Western blot analysis of Ad-LacZ or N4/int-3 endothelial cells using anti-HA antibody. (C) Activation of a CSL luciferase reporter in endothelial cells expressing N4/int-3, represented as fold induction in Ad–N4/int-3 cells relative to Ad-LacZ. Data are an average of 3 independent experiments. (D) qRT-PCR of Tie and VEGFRs in Ad-infected endothelial cells. Values were normalized to β-actin qRT-PCR. Data of 3 independent qRT-PCR analyses are represented as fold induction in Ad–N4/int-3 cells relative to Ad-LacZ. (E) Western blot analysis of cell surface proteins isolated from Ad-LacZ and Ad–N4/int-3 endothelial cells immunoblotted with anti–VEGFR-3 antibody and total cell lysates immunoblotted with anti–α-tubulin antibody. (F) Western blot analysis of cell surface proteins (CSPs) isolated from Ad-LacZ–, Ad–N4/int-3–, and Ad-N1IC–infected endothelial cells immunoblotted with antibodies against VEGFR-3 and α-tubulin. Total cell lysates (TCLs) immunoblotted with antibodies against the HA epitope of N4/int-3 (12CA5), N1IC, and α-tubulin. (G and H) VEGFR-3, Hey1, and Hey2 qRT-PCR of Jagged1/Notch4 (J1/N4) or Dll4/Notch4 (Dll4/N4) HUVEC cocultures, respectively. Values were normalized by β-actin qRT-PCR and are presented as relative values. (I) VEGFR-2 and VEGFR-3 FACS of Ad-LacZ, Ad-N1IC, and Ad–N4/int-3 BECs.

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques: Western Blot, Activation Assay, Luciferase, Expressing, Quantitative RT-PCR, Infection, Isolation

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Viability of E9.5 and P21 mice from Notch1+/– and VEGFR-3+/– crosses

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques:

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Notch1+/– (A and D), VEGFR-3+/– (B and E), and Notch1+/–;VEGFR-3+/– (C and F) E9.5 embryos were immunostained for PECAM. (D–F) The dorsal region of embryos was enlarged to visualize the dorsal aorta (blue arrowheads), atria of the primary heart tube (green arrowheads), and intersomitic vessels. E9.5 VEGFR-3+/– (G and I) and Notch1+/–;VEGFR-3+/– (H and J) embryos were β-gal stained to visualize the VEGFR-3–positive vasculature. (I and J) The cranial region of the embryos was enlarged to demonstrate the decrease in VEGFR-3 expression (red asterisks) and reduction of VEGFR-3–positive vessel density (yellow arrowheads). Original magnification, ×5 (A–C, G, and H); ×15 (D–F); ×10 (I and J).

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques: Staining, Expressing

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Murine P4 dermal sections immunostained with PECAM (A and J), VEGFR-3 (D and M), LYVE-1 (G and P), Notch1 (B, E, and H), or Notch4 (K, N, and Q) antibodies. Notch1 is coexpressed with PECAM (C), VEGFR-3 (F), and LYVE-1 (I). Notch4 is coexpressed with PECAM (L), VEGFR-3 (O), and LYVE-1 (R). (M–R) White arrowheads highlight Notch4 staining in the dermal lymphatic endothelium. Original magnification, ×12.5 (A–C and J–L); ×40 (D–I and M–R).

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques: Staining

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Human IMC immunostained with VEGFR-3 (B and E), LYVE-1 (H and L), podoplanin (P), Notch1 (A and G), Notch4 (D and K), or activated Notch1 (N1Val) (O) antibodies. Notch1 (C) and Notch4 (F) are coexpressed with VEGFR-3 in the extratumoral IMC vessels. Notch1 (I) and Notch4 (M) are coexpressed with LYVE-1 in the extratumoral lymphatics. J and N represent magnified view of boxed areas in I and M, showing lymphatic vessels. (N) White arrowhead indicates Notch4-positive LEC. Yellow arrowheads highlight Notch4-positive non-endothelial cells adjacent to the lymphatic endothelium. (Q) The activated Notch1 peptide is localized to the LEC nuclei. White arrowheads highlight positive nuclei, and yellow arrowhead indicates a negative nucleus. Original magnification, ×50 (A–F and O–Q); ×40 (G–I and K–M); ×120 (J and N).

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Inhibition of the Notch pathway aggravates DSS-induced colitis. CT: control, LY411,575 (Notch inhibitor): 10 mg/kg LY411,575 gavage, DSS: 2.5% DSS water, DSS-LY411,575: LY411,575 was intragastric administrated at the last 4 days of DSS feeding (once per day). (A) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (B) Serum FD-4 concentration in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (C) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (D) Representative colonic H&E staining image of each group (scale bar = 50 μm). (E) Representative colonic IF staining image of claudin-1 ( green ) and claudin-3 ( green ) (scale bar = 25 μm). (F) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) (scale bar = 25 μm). Quantification of N1ICD and Hes1 were determined by the ratio of positive staining cells per crypt. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Inhibition, Control, Concentration Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Notch pathway is inhibited in the colon under VDd or VDR KO conditions. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO (A) and VDd (B) mice (scale bar = 25 μm). (C, D) Real-time PCR analysis of the gene expression of Notch ligands, receptors, and effectors in the colon in VDR KO (C) and VDd (D) mice. Expression levels (VDR KO vs. WT or VDd vs. CT) were compared using paired t -test. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR modulates the Notch pathway in colitis. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDd-DSS and respective control colitis mice (scale bar = 25 μm). (C, D) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO-DSS and respective control colitis mice. To activate VDR, mice were administered paricalcitol (PAR) 7 days before drinking DSS water and were sustained until sacrifice. (E) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (F) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (G) Representative colonic H&E staining image of each group (scale bar = 50 μm). (H) Representative colonic IF staining image of claudin-1 ( green ), claudin-3 ( green ), and Hes1 ( red ) (scale bar = 25 μm). Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling regulates the Notch pathway and TJs in Caco-2 cells. TNF-α (100 ng/mL) was added to mimic inflammatory stimulation, and 100 nM PAR was used to activate VDR; the solvate was added as control (CT). Specific siRNA of VDR (SiVDR) was used to downregulate VDR, negative control (NC) sequence was used as control (scale bar = 10 μm). (A) Representative IF staining image of claudin-1 ( green ) and claudin-3 ( green ) in Caco-2 cells treated with or without PAR before TNF-α intervention. (B) Representative IF staining image of N1ICD ( red ) and Hes1 ( red ) in Caco-2 cells with or without VDR downregulation before TNF-α intervention. (C) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in Caco-2 cells after VDR downregulation.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Negative Control, Sequencing, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR-mediated TJ protection is partly dependent on the Notch pathway. LY411,575 (1 μM) was used to inhibit the Notch pathway in Caco-2 cells, and 100 nM PAR was used to activate VDR. The solvate was added as control (CT). Representative IF staining image of N1ICD ( red ), Hes1 ( red ), claudin-1 ( green ), and claudin-3 ( green ) in Caco-2 cells treated with or without LY411,575 before PAR intervention (scale bar = 10 μm).

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling positively regulates the Notch-1/Hes1 pathway. (A) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in cultured intestinal organoids (scale bar = 10 μm). (B) Western blot analysis of N1ICD and Hes1 proteins in cultured intestinal organoids from WT and VDR KO mice. (C) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in cultured intestinal organoids from WT and VDR KO mice. (D) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after TNF-α intervention. (E, F) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after downregulation of VDR with siVDR (E) or activation of VDR with PAR (F) . Data are presented as the mean ± SEM from three separate experiments, the statistical analysis performed with paired t -test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR positively regulates Notch-1 transcription. (A) Western blot analysis of VDR, Notch-1, N1ICD, and Hes1 in Caco-2 cells after siVDR and PAR intervention. (B) Graphs of the predicted VDR binding region in the Notch-1 promoter. (C) Dual-luciferase reporter assay of VDR to Notch-1 promoter. Notch-1 promoter (Notch-1) and negative control (CON) were constructed in a GV238 vector. The VDR cDNA and vector (pcDNA3.1) were linked to upregulate VDR (pVDR). Data are presented as the mean ± SEM from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Western Blot, Binding Assay, Luciferase, Reporter Assay, Negative Control, Construct, Plasmid Preparation