202399 jag1 fc r d systems  (R&D Systems)

Journal: BDJ Open

Article Title: MicroRNA expression in JAG1/Notch-activated periodontal ligament stem cells

doi: 10.1038/s41405-024-00232-5

Figure Lengend Snippet: Representative histogram indicating expression of MSCs markers as evaluated by flow cytometry ( a ). Multilineage differentiation of the osteo/odontogenic and adipogenic lineages examined by alizarin red s staining and oil red o staining ( b ). Cells were seeded on Jagged1 immobilized surface and maintained in osteo/odontogenic medium. Cells on hFc-immobilized surfaces were used as controls. Osteo/odontogenic differentiation was evaluated in vitro using alizarin red s staining ( c ).

Article Snippet: Briefly, recombinant protein G (Sigma-Aldrich, USA) was incubated in a tissue culture plate at a concentration of 50 μg/mL for 16 h. The surface was then incubated with 10 mg/mL of BSA (Sigma-Aldrich, USA) for 2 h followed by incubation with Jagged1/Fc (R&D systems, USA) for 2 h. An equal volume of human IgG, the Fc fragment (Sigma-Aldrich, USA), was used as a control.

Techniques: Expressing, Flow Cytometry, Staining, In Vitro

Journal: BDJ Open

Article Title: MicroRNA expression in JAG1/Notch-activated periodontal ligament stem cells

doi: 10.1038/s41405-024-00232-5

Figure Lengend Snippet: Heatmap of significantly alter miRNAs in Jagged1-treated PDLSCs ( a ). Venn diagram of predicted target genes of the significantly altered miRNAs ( b ).

Article Snippet: Briefly, recombinant protein G (Sigma-Aldrich, USA) was incubated in a tissue culture plate at a concentration of 50 μg/mL for 16 h. The surface was then incubated with 10 mg/mL of BSA (Sigma-Aldrich, USA) for 2 h followed by incubation with Jagged1/Fc (R&D systems, USA) for 2 h. An equal volume of human IgG, the Fc fragment (Sigma-Aldrich, USA), was used as a control.

Techniques:

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) Western blot analysis of mock- or R-Ras38V-transduced EC lysate from confluent culture. Notch1 intercellular domain (N1ICD) was detected by an antibody specific for it. Since N3ICD-specific antibody is unavailable, it was detected by anti-Notch3 antibody based on the molecular weight of N3ICD. Notch3 [FL], full-length Notch3; Notch3 [TM], transmembrane fragment of cleaved Notch3; N3ICD, Notch3 intercellular domain. (B) Quantification of (A). N = 3 (Notch1), N = 4 (Jagged2), N = 5 (Dll4 and all Notch3 species), N = 6 (Jagged1 and N1ICD). Data are represented as the mean ± SEM. See also . (C) Immunofluorescence staining of confluent EC culture for N1ICD, N3ICD, and Jaggged1 with representative pictures and the quantification of fluorescence intensity presented as relative values (RU). DAPI nuclear staining is blue. Data are represented as the mean ± SEM. N = 3 wells. Pictures of two or three different areas were analyzed for each well. (D) Jagged1 expression in lung capillary ECs was analyzed in cdh5 -Cre; Rras f/f mice ( Rras ΔEC ) and Rras f/f wild-type control mice (WT) by immunostaining. Jagged1 intensity within the CD31 + area was quantified and normalized to the total CD31 + area. N = 3 mice. Pictures of three different areas of each lung were analyzed. Data are represented as the mean ± SEM. Scale bar, 50 μm. (E) Western blot analysis of small interfering RNA (siRNA) control (siCont) and R-Ras-silenced EC (siRRas). See also . (F) Quantification of (E). N = 3. Data are represented as the mean ± SEM. (G) Jagged1 and N1ICD levels in the control or R-Ras-silenced ECs were determined by immunofluorescence. The graphs present the fluorescence intensity as relative values (RU). DAPI nuclear staining is blue. N = 3 wells, one or two pictures were analyzed per well. Data are represented as the mean ± SEM. See also – . (H) Jagged1 was silenced (siJagged1) in the mock control or R-Ras38V-expressing ECs, and the N1ICD levels in these cells were analyzed by immunofluorescence (red). N = 2. (I) Jagged1 was silenced in the confluent culture of parental ECs, and Notch activation was analyzed by western blot. The graph presents Notch3 in relative values. N = 3. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, n.s., not significant.

Article Snippet: To study the effect of Notch activation by Jagged1, 3.5 cm plates were coated with 5 μg/ml rhJagged1 (#1277-JG, R&D Systems, MN) for 2 hours at 37°C.

Techniques: Western Blot, Molecular Weight, Immunofluorescence, Staining, Fluorescence, Expressing, Control, Immunostaining, Small Interfering RNA, Activation Assay

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) MTT assay of mock and R-Ras38V-expressing ECs. N = 10 wells. Data are represented as the mean ± SEM. (B) BrdU incorporation and Ki-67 staining of mock and R-Ras38V-expressing ECs to assess cell cycling. N = 3 wells, three pictures analyzed per well. Data are represented as the mean ± SEM. See also . (C) In vitro endothelial sprouting assay. Endothelial sprouting of R-Ras-silenced ECs were analyzed in 3D fibrin gel culture at 24, 48, and 72 h. Silencing of R-Ras exacerbated the vessel sprouting. (D) Ki-67 staining of whole-mount neonatal retinal vasculature (day 6). ECs are visualized by IB4 staining. Ki-67 intensity in the IB4 + area was quantified and normalized to the IB4 + area. N = 4 retinas, two or three pictures were analyzed for each retina. Data are represented as the mean ± SEM. (E) Ki-67 staining of proliferating ECs in the coculture system. Green-labeled mock- (EC mock ) or R-Ras38V-transduced ECs (EC RRas38V ) were transfected with Jag1 (siJag1) or control siRNA for 15 h and then cocultured with non-transduced unlabeled parental ECs (EC) for an additional 48 h. DAPI was used for nuclear staining (blue). (F) Migration of R-Ras- or Jagged1-silenced ECs was analyzed by a scratch-wound assay and quantified as the percentage of closure of the wound by migrated ECs in 24 h. N = 4 culture dishes. Data are represented as the mean ± SEM. (G) Transwell migration assay of the control and R-Ras-silenced ECs. Cells that migrated to the bottom side of the Transwell inserts were stained with 0.5% crystal violet and quantified. N = 6 Transwell inserts. Data are represented as the mean ± SEM. See also . (H) Transwell migration of ECs treated with or without 10 μM DAPT. N = 3 Transwell inserts. Data are represented as the mean ± SEM.

Article Snippet: To study the effect of Notch activation by Jagged1, 3.5 cm plates were coated with 5 μg/ml rhJagged1 (#1277-JG, R&D Systems, MN) for 2 hours at 37°C.

Techniques: MTT Assay, Expressing, BrdU Incorporation Assay, Staining, In Vitro, Labeling, Transfection, Control, Migration, Scratch Wound Assay Assay, Transwell Migration Assay

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) Cocultures of EC mock or EC RRas38V (green) with unlabeled, non-transduced parental ECs (EC) were stained for VE-cadherin to analyze the integrity of the adherens junctions between ECs. DAPI stain is in blue. Scale bars, 10 μm. N = 3. (B) Unlabeled parental ECs were cocultured with green-fluorescence-labeled R-Ras38V-expressing ECs with or without Jagged1 silencing (EC RRas38V/siJag1 or EC RRas38V/siCont ), and the adherens junctions between the ECs were examined by VE-cadherin staining. Scale bars, 50 μm. N = 3. (C and D) Western blot of control and Jagged1-silenced (C) or R-Ras-silenced (D) ECs. VE-cadherin protein levels were quantified and normalized to β-actin. VE-cad, VE-cadherin. Data are represented as the mean ± SEM. N = 3. See also and . (E) The protein levels of UNC5b (N = 6) and protein (N = 10) and mRNA levels (N = 3) of VE-cadherin (CDH5) in mock- or R-Ras38V-transduced ECs were determined by western blot and RT-qPCR. Data are represented as the mean ± SEM. (F) UNC5b and VE-cadherin western blot of mock- or R-Ras38V-transduced ECs treated with or without 10 μM DAPT. N = 3. (G) VE-cadherin immunostaining (magenta) of mock- or R-Ras38V-transduced ECs with or without UNC5b silencing. Surface plot images show the fluorescence intensity of pixels in color scale. Data are represented as the mean ± SEM. N = 3 wells, three pictures analyzed for each. See also – . (H) EC RRas38V (green) were cocultured with UNC5b-silenced or control ECs (unlabeled). The coculture was then stained for VE-cadherin (magenta). Top: white arrows indicate a strong accumulation of VE-cadherin at the cell membrane of control ECs adjacent to EC RRas38V . Bottom: yellow arrows indicate the loss of VE-cadherin at the cell-cell junctions in neighboring ECs in which UNC5B was silenced. Representative images of three independent experiments are shown. Scale bars, 10 μm. (I) VE-cadherin and UNC5b staining of whole-mount neonatal retinal vasculature (day 6). ECs are visualized by IB4 staining. VE-cadherin or UNC5b intensity in the IB4 + area was quantified and normalized to the IB4 + area. N = 8 retinas, five to eight pictures per retina. Data are represented as the mean ± SEM.

Article Snippet: To study the effect of Notch activation by Jagged1, 3.5 cm plates were coated with 5 μg/ml rhJagged1 (#1277-JG, R&D Systems, MN) for 2 hours at 37°C.

Techniques: Staining, Fluorescence, Labeling, Expressing, Western Blot, Control, Quantitative RT-PCR, Immunostaining, Membrane

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) EC mock or EC RRas38V with or without Jagged1 silencing were cocultured with parental ECs at a 1:3 ratio. Confluent monolayers of various coculture combinations were examined for endothelial permeability by dextran leakage. Single culture of EC RRas38V was included for comparison with the cocultures. N = 3. Data are represented as the mean ± SEM. (B) Extravascular fibrinogen in the lung was analyzed by immunostaining in 3D-reconstructed confocal images. Fluorescence intensity was normalized to the total CD31 + area. Unc5b staining intensity within the CD31 + area was quantified and normalized to the total CD31 + area. The white square insets of 72 × 72 μm are shown in higher magnification in the lower images. N = 3 mice, seven or eight pictures analyzed for each lung. Data are represented as the mean ± SEM. (C) Vascular permeability in adult retina was examined by perfusion with a biotinylation reagent (sulfo-NHS-biotin) followed by detection with streptavidin and IB4 staining. The retinal areas indicated by the squares are magnified on the right. The extravascular sulfo-NHS-biotin fluorescence intensity was quantified from the images and normalized to the total IB4 + area. N = 6 retinas. Data are represented as the mean ± SEM. See also . (D) Fibrinogen staining (red) to assess plasma leakage in the hippocampus. The intensity of extravascular fibrinogen was normalized to the total CD31 + area. N = 3 mice, six or more pictures analyzed for each brain. Data are represented as the mean ± SEM. (E) Hey1, Unc5b, or VE-cadherin intensity within the CD31 + area of the hippocampus was quantified and normalized to the CD31 + area. DAPI for nuclear staining is blue. Scale bars, 50 μm. N = 3 mice, five or more pictures analyzed for each brain. Data are represented as the mean ± SEM. See also . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To study the effect of Notch activation by Jagged1, 3.5 cm plates were coated with 5 μg/ml rhJagged1 (#1277-JG, R&D Systems, MN) for 2 hours at 37°C.

Techniques: Permeability, Comparison, Immunostaining, Fluorescence, Staining

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) The gene expression levels in HUVECs were compared between Akt isoforms by RNA-seq. N = 3. Data are represented as the mean ± SD. (B and C) The protein level of each Akt isoform (N = 3–5) and Ser473 phosphorylation of all Akts were determined in R-Ras38V-transduced (B) or R-Ras-silenced (C) ECs by western blot. For quantification, pAkt Ser473 was normalized to the total Akt. Data are represented as the mean ± SEM. (D) Phosphorylation of each Akt isoform was determined in mock- or R-Ras38V-transduced ECs by immunoprecipitation of the total pAkt Ser473 followed by western blot of each isoform. (E) Each Akt isoform was silenced in mock- or R-Ras38V-transduced ECs and the protein levels of Jagged1 and p21 were determined in these cells. (F) The Akt3 isoform was silenced in R-Ras38V-transduced ECs, and Notch1 and Notch3 activation as well as the protein levels of Jagged1, p21, VE-cadherin, and UNC5b were determined. Data are represented as the mean ± SEM. (G) Akt3 was silenced in mock- (EC mock ) or R-Ras38V-transduced ECs (EC RRas38V ) that were cocultured with parental ECs (EC), and Jagged1 expression was evaluated. *p < 0.05, **p < 0.01; n.s., not significant.

Article Snippet: To study the effect of Notch activation by Jagged1, 3.5 cm plates were coated with 5 μg/ml rhJagged1 (#1277-JG, R&D Systems, MN) for 2 hours at 37°C.

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Immunoprecipitation, Activation Assay

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) Migration of R-Ras-, Akt3-, or Jagged1-silenced ECs was analyzed by a scratch-wound assay and quantified as the percentage of closure of the wound in 24 h. N = 3 culture dishes. Data are represented as the mean ± SEM. (B) si-knockdown efficacy on the scratch assay in (A) analyzed by western blot. (C) Akt3 was silenced in mock- or R-Ras38V-transduced ECs, and the migration of these cells was analyzed by Transwell migration assay. Cell migration is shown in relative values. N = 3 Transwell inserts, five or more pictures analyzed for each. Data are represented as the mean ± SEM. (D) Jagged1 was silenced in mock- or R-Ras38V-transduced ECs and an MTT assay was performed to analyze cell proliferation. N = 7 wells. Data are represented as the mean ± SEM. (E) EC mock or EC RRas38V with and without Akt3 silencing (green) were cocultured with unlabeled parental ECs at a 1:3 ratio. VE-cadherin and Ki-67 were stained and imaged 48 h later. Ki-67 + cells in the transduced EC population (green) and those in the parental (neighboring) EC population were quantified. Data are represented as the mean ± SEM in violin plots. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. N = 4 wells, multiple pictures were analyzed for each well.

Article Snippet: To study the effect of Notch activation by Jagged1, 3.5 cm plates were coated with 5 μg/ml rhJagged1 (#1277-JG, R&D Systems, MN) for 2 hours at 37°C.

Techniques: Migration, Scratch Wound Assay Assay, Knockdown, Wound Healing Assay, Western Blot, Transwell Migration Assay, MTT Assay, Staining

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet:

Article Snippet: To study the effect of Notch activation by Jagged1, 3.5 cm plates were coated with 5 μg/ml rhJagged1 (#1277-JG, R&D Systems, MN) for 2 hours at 37°C.

Techniques: Virus, Control, Expressing, Plasmid Preparation, Recombinant, Isolation, MTT Assay, Western Blot, Software