Journal: Journal of translational autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells.

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Fig. 3. Immunofluorescent antibody staining in naïve and treated HUVECs. AK. The mixture of APS total IgG and β2GPI induces a pronounced increase of the protein levels of the proinflammatory cytokines IL-6, IL-8 as well the transcription factor NF-κB1 and cell adhesion molecules Tissue Factor, ICAM-1, VCAM-1, Eselectin, P- selectin and TGFR1. There was no significant difference for the TGFR1 molecule (3 J). Visual analysis revealed that all the inflammatory mediators and adhesion molecules presented statistically significant difference between the untreated and treated endothelial cells (3 K).

Article Snippet: Coverslips were incubated overnight at 4 ◦C with primary antibodies against IL-6 (5 μg/ ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSBPA023451LA01HU, Cusabio).

Techniques: Staining

Journal: Journal of translational autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells.

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Fig. 5. Immunofluorescent antibody staining in placenta biopsies from APS patients and healthy women. A-K Placenta biopsies derived from APS patients as well as Healthy Donors show increased signal intensity for IL-6, IL-8, NF-κB1, ICAM1, VCAM-1, E-selectin, P-selectin, TGF-β2, and TGFR1 (5A-5D, 5F-5J). Slight difference in fluorescence intensity between HD and APS patient was observed for Tissue Factor (5E). Increased signal intensity was observed as well for the TNF-α molecule in the APS placenta biopsies (5 K).

Article Snippet: Coverslips were incubated overnight at 4 ◦C with primary antibodies against IL-6 (5 μg/ ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSBPA023451LA01HU, Cusabio).

Techniques: Staining, Derivative Assay, Fluorescence

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Primer sequences.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques:

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Mutation in exon 3 of TGFBR1 isolated from a patient with LDS and aggressive periodontitis diminishes the response to TGF-β (G188V mutation). (A) Location of the mutation in exon 3 of TGFBR1 . (B) HEK293 cells were transfected with a vector alone, wildtype mouse Tgfbr1 ( Tgfbr1 W T ), or mouse Tgfbr1 with the G188V mutation ( Tgfbr1 G 188V ). Lysates of transfected cells were subjected to western blot analysis using an anti-TGFBR1 antibody. (C) TGF-β-induced promoter activity was assessed by the dual luciferase reporter assay system. Values represent the mean ± SD in triplicate assays. ∗ P < 0.05, compared with the indicated TGF-β concentration.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques: Mutagenesis, Isolation, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Luciferase, Reporter Assay, Concentration Assay

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Generation of knock-in mice with the Tgfbr1 G188V mutation. (A) Structure of the Tgfbr1 G188V mutant allele. (B) No clear differences in 6-week-old Tgfbr1 G 188V/+ mice. (C) PCR genotyping of genomic DNA using tail specimens collected from Tgfbr1 G 188V/+ and WT mice. (D) Embryos of WT, Tgfbr1 G 188V/+ , and Tgfbr1 G 188V/G188V at embryonic day 14.5.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques: Knock-In, Mutagenesis

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Number of mice with each genotype.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques:

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Tgfbr1 G 188V/+ mice recapitulate vascular LDS phenotypes. (A) Kaplan–Meier survival curve showing a diminishing lifespan of Tgfbr1 G 188V/+ mice. ∗ P < 0.05. (B) Representative aortic wall sections of Tgfbr1 G 188V/+ mice at 24 weeks of age stained with EVG to identify elastin fibers. Scale bar: 25 μm. Arrows show elastic fiber distortion. Arrowheads show elastic fiber fragmentation. (C) Analysis of TGF-β-related genes in aortic tissues derived from wildtype and Tgfbr1 G 188V/+ mice ( n = 4). Data represent means ± SD in triplicate assays. (D) Serpine1 gene expression in MEFs derived from wildtype, Tgfbr1 G 188V/+ , and Tgfbr1 G 188V/G188V embryos at embryonic day 13.5 stimulated with TGF-β (0–5 ng/mL). Data represent means ± SD in triplicate assays. ∗ P < 0.05, compared with the indicated TGF-β concentration. (E) Phospho-Smad2 (pSmad2) in MEFs derived from wildtype, Tgfbr1 G 188V/+ , and Tgfbr1 G 188V/G188V embryos at embryonic day 13.5 stimulated with TGF-β (0–10 ng/mL). Quantitative western blot analysis is shown as the ratios of the intensities of phospho-Smad2 to Smad2.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques: Staining, Derivative Assay, Gene Expression, Concentration Assay, Western Blot

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Analysis of phenotypes in periodontal tissues of Tgfbr1 G 188V/+ mice. (A) Representative images from micro-CT analysis of maxillary alveolar bones in Tgfbr1 G 188V/+ mice. Scale bar: 1 mm. (B) Alveolar bone resorption was measured in the root surface area between the alveolar apex from the cementoenamel junction (red line in ). Data represent means ± SD. Six-week-old wildtype: n = 6, 6-week-old Tgfbr1 G 188V/+ : n = 8, 24-week-old wildtype: n = 20, and 24-week-old Tgfbr1 G 188V/+ : n = 14. (C) Representative images of HE staining of the periodontium from WT and Tgfbr1 G 188V/+ mice at 6 and 24 weeks of age. AB, alveolar bone; PDL, periodontal ligament; D, dentin. Scale bar: 50 μm. (D) Analysis of TGF-β-related gene expression in periodontal tissues derived from wildtype and Tgfbr1 G 188V/+ mice ( n = 4). Data represent means ± SD in triplicate assays. ∗ P < 0.05, ∗∗ P < 0.01, compared with wildtype.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques: Micro-CT, Staining, Gene Expression, Derivative Assay

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Oral infection with P. gingivalis induces alveolar bone resorption in Tgfbr1 G 188V/ + mice. (A) Representative images from micro-CT analysis of maxillary alveolar bones in P. gingivalis -infected WT and Tgfbr1 G 188V/+ mice. Scale bar: 1 mm. (B) Alveolar bone resorption was measured in the root surface area between the alveolar apex from the cementoenamel junction (red line in A ). Data represent means ± SD in triplicate assays. ∗∗ P < 0.01. Wildtype: n = 11 in vehicle, n = 6 in P. gingivalis infection. (C) Representative images of HE staining of the periodontium from P. gingivalis -infected wildtype and Tgfbr1 G 188V/+ mice. Right panel shows a high magnification images of the rectangular area indicated in the left panel. Scale bar: 200 μm. Arrowheads indicate multinucleated cells. AB, alveolar bone; PDL, periodontal ligament; D, dentin. (D) Representative images of TRAP-stained periodontium from P. gingivalis -infected wildtype and Tgfbr1 G 188V/+ mice. Right panel shows a high magnification image of the rectangular area indicated in the left panel. Scale bar: 200 μm. Arrowheads indicate TRAP-positive multinucleated cells. (E) Quantification of TRAP-positive cell numbers in each group. TRAP-positive cell numbers on the surface of alveolar bone were counted and divided by the length of the alveolar bone (red line in D ). Data represent means ± SD in wildtype: n = 3 in vehicle, n = 5 in P. gingivalis infection. Tgfbr1 G 188V/+ : n = 4 in vehicle, n = 6 in P. gingivalis infection. Vehicle: sham control, P.g , P. gingivalis infection, AB, alveolar bone; D, dentin; PDL, periodontal ligament. ∗ P < 0.05, compared with Vehicle.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques: Infection, Micro-CT, Staining, Control

Journal: Frontiers in Physiology

Article Title: Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

doi: 10.3389/fphys.2021.715687

Figure Lengend Snippet: Upregulation of inflammatory cytokine mRNA expression in peritoneal macrophages from Tgfbr1 G 188V/+ mice. Macrophages were harvested from the peritoneal cavity of 8-week-old male WT and Tgfbr1 G 188V/+ mice at 3 days after injection of thioglycolate. The peritoneal macrophages were stimulated with 1 μg/ml P. gingivalis LPS for the indicated times and then total RNA was isolated for quantitative PCR analysis. Data represent means ± SD in triplicate assays. ∗∗ P < 0.01, compared with wildtype.

Article Snippet: Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Injection, Isolation, Real-time Polymerase Chain Reaction