Journal: Biology of Sex Differences

Article Title: Sex differences in disease severity and immune responses in murine and human inflammatory arthritis

doi: 10.1186/s13293-026-00840-w

Figure Lengend Snippet: Male CIA mice exhibit higher disease severity compared to females. Male and female CIA and saline control mice were monitored for disease severity and assigned clinical scores from day 21 after the first CII challenge until the end of experiment (day 29). (A) Line graphs representing the mean clinical scores of mice starting from day 1 to day 29. On day 29 after the first CII challenge, mice were euthanized by cardiac puncture under anesthesia for blood and serum collection and storage at −80 °C. Serum concentrations of (B) anti-mouse collagen type II antibodies (left panel) and anti-bovine collagen type II antibodies (right panel) were analyzed by ELISA. N = 5 mice per group. One of two independent experiments. Simple linear regression analysis was performed to determine the statistical difference between the lines. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test was used to determine the statistical significance between the groups. (*/ # p ≤ 0.05, ** p ≤ 0.005 and ***/ ### p ≤ 0.0005). In Fig. 1A, # significance of comparisons between CIA and saline control mice; *significance of comparisons between sexes of CIA mice

Article Snippet: Serum levels of mouse anti-collagen antibodies (autoantibodies) and bovine anti-collagen antibodies (antibodies to the immunizing antigen) were determined by ELISA using a Mouse Anti-mouse Type II Collagen IgG Antibody Assay Kit and Mouse Anti-Bovine Type II Collagen IgG Antibody Assay Kit, respectively, according to the manufacture’s protocol (Chondrex Inc. WA, USA).

Techniques: Saline, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific reports

Article Title: Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

doi: 10.1038/s41598-024-59713-0

Figure Lengend Snippet: Figure 7. Immunocytochemical features of gingival connective tissues in the gingiva of RNS variants. (A) In normal gingiva, POSTN is localized in a loose extracellular network around blood vessels (arrowheads; bv) whereas in both RNS-1 (B) and RNS-2 (C) mutants POSTN extracellular expression is localized along thick and disorganized fibers in the connective tissue (ct). (D) In normal gingiva, Collagen 5 alpha-1 (green; Col5A1) is secreted and deposited along collagen 1 fibers and Vimentin (red; Vim) is expressed by endothelial cells. (E,F) Increased expression of Col5A1 is detected in the connective tissue (ct) of RNS-1 (E) and RNS-2 (F) mutants. Vim is normally localized in endothelial cells and abnormally in fibroblasts (F). (G) Alpha-SMA (α-SMA) is a specific marker of contractile endothelial cells of bv. (H,I) In RNS mutants, α-SMA is also expressed by fibroblasts in ct. Note the presence of inflammatory infiltrates (i) in RNS-1 gingiva. (J) In normal gingiva, Fibroblast-specific protein 1 (green; FSP1) is expressed by gingival fibroblasts (green arrow). Osteonectin (red; SPARC) is expressed in discrete spots by fibroblasts (red arrowhead) and endothelial cells. (K,L) FSP-1 is normally expressed in ct. SPARC expression and SPARC deposition along fibers are dramatically increased in RNS mutant connective tissues. (M) Fibronectin (green; FN) is specifically expressed by fibroblasts. Phospho- SMAD3 (red; pSMAD3) is mainly localized in nuclei of vascular cells (arrowhead). (N,O) In RNS-derived gingiva FN expression is dramatically decreased. pSMAD3 expression is dramatically increased in both vascular cells (arrowhead) and fibroblasts (arrows) nuclei. Scale bars: (A–C), (G–L) = 100 μm; (D–F), (M–O) = 30 μm.

Article Snippet: Sections were incubated for 2 h at room temperature or overnight at 4 °C with primary antibodies; rabbit anti-collagen V alpha-1 (1/1000; NBP-38162; Novus biologicals), rabbit anti-FAM20A (1/250; OACD03385; Aviva), rabbit anti-Fam20C (1/250; OAAB01003; Aviva), rabbit antiPeriostin (1/500; ab14041; Abcam), rabbit anti-phospho-SMAD3 (1/500; ab52903; Abcam), rabbit anti-SPARC (1/2000; ab255733; ABCAM), mouse anti-alpha Smooth Muscle Actin (1/1000; 1A4; ab7817; Abcam), mouse anti-Fibronectin (1/1000; P1H11; DSHB), mouse anti-FSP1 (1/2000; ab218512; ABCAM), mouse anti-Vimentin (1/1000; RV203; NBP-19767; Novus biologicals) and rat anti-Procollagen type I (1/500; M-58; ab64409; Abcam), mouse anti-Ubiquitin (UCHL1; 1/400; 66230-1-Ig; Proteintech), goat anti-Lamin B1(1/200; sc6216), Concanavalin A-Alexa 633 conjugate (70 mg/ml; 2616059; Thermo Fischer).

Techniques: Expressing, Marker, Mutagenesis, Derivative Assay

Journal: Scientific reports

Article Title: Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

doi: 10.1038/s41598-024-59713-0

Figure Lengend Snippet: Figure 8. SMAD3 activation in untreated RNS GFs and treated control and RNS GFs. Immunocytochemical staining of control (A,D) and RNS (B,C,E,F) GFs cultured without TGFβ1 (A–C) or with 5 ng/ml TGFβ1 (D–F) for 6 h. Cells were fluorescently labeled for p-SMAD3 (green), nuclei (blue), and the specific F-actin marker, phalloidin (red). Co-localization of p-SMAD3 and nuclei indicated nuclear translocation of p-SMAD3 (green arrow). (A) In control untreated GFs, p-SMAD3 immunoreactivity is detected at low levels in some nuclei. (B,C) In RNS untreated GFs, p-SMAD3 was increased in intensity in all nuclei. (D–F) TGFβ1 induced an increase in p-SMAD3 nuclei in normal (D) and mutant GFs (E,F). (G) Western blot were performed on cell lysates. P-YAP (Ser 397) protein levels were decreased in RNS GFs without or with TGFβ1 compared to Control. TAZ protein levels were increased in RNS GFs compared to normal GFs. Densitometric analysis of Phospho-YAP and TAZ bands normalized to corresponding GAPDH bands. Data represent mean fold change in band intensity ± s.d. relative to GAPDH of 3 independent experiments in triplicates. Data were analyzed by one-way ANOVA with Bonferroni multiple comparisons test (**p < 0.01, ***p < 0.001). (H) Western blots were performed on cell lysates. Alpha-SMA protein levels were increased in controls GFs cultured with TGFβ1. Alpha-SMA protein levels were increased in RNS GFs cultured without or with TGFβ1 compared to control. Densitometric analysis of a-SMA bands normalized to corresponding GAPDH levels. Scale bars: (A-F) = 20 µm.

Article Snippet: Sections were incubated for 2 h at room temperature or overnight at 4 °C with primary antibodies; rabbit anti-collagen V alpha-1 (1/1000; NBP-38162; Novus biologicals), rabbit anti-FAM20A (1/250; OACD03385; Aviva), rabbit anti-Fam20C (1/250; OAAB01003; Aviva), rabbit antiPeriostin (1/500; ab14041; Abcam), rabbit anti-phospho-SMAD3 (1/500; ab52903; Abcam), rabbit anti-SPARC (1/2000; ab255733; ABCAM), mouse anti-alpha Smooth Muscle Actin (1/1000; 1A4; ab7817; Abcam), mouse anti-Fibronectin (1/1000; P1H11; DSHB), mouse anti-FSP1 (1/2000; ab218512; ABCAM), mouse anti-Vimentin (1/1000; RV203; NBP-19767; Novus biologicals) and rat anti-Procollagen type I (1/500; M-58; ab64409; Abcam), mouse anti-Ubiquitin (UCHL1; 1/400; 66230-1-Ig; Proteintech), goat anti-Lamin B1(1/200; sc6216), Concanavalin A-Alexa 633 conjugate (70 mg/ml; 2616059; Thermo Fischer).

Techniques: Activation Assay, Control, Staining, Cell Culture, Labeling, Marker, Translocation Assay, Mutagenesis, Western Blot