spon1  (Cusabio)

Journal: Dentistry Journal

Article Title: Bone Morphogenetic Protein 7 Promotes the Differentiation of Periodontal Ligament Fibroblasts into F-Spondin-Expressing Cementoblast-like Cells During Root Canal Treatment—An In Vivo Rat Pulpectomy Model and In Vitro Human Fibroblast Study

doi: 10.3390/dj13110494

Figure Lengend Snippet: Immunohistochemical staining of Spon1 of the apical area of the mesial root after root canal treatment. The control group was filled with PGA only, and the experimental group was filled with PGA and 50 µg/mL BMP-7. ( A , B ) Control group at 14 days postoperatively. ( C , D ) BMP-7 group at 14 days postoperatively. ( E , F ) Control group at 28 days postoperatively. ( G , H ) BMP-7 group at 28 days postoperatively. ( B , D , F , H ) Expanded images of the boxed areas indicated in ( A , C , E , G ), respectively. ( n = 5) ( A ) A few Spon1-positive areas were observed. ( B ) Spon1-positive areas were observed compared to the control group. ( C ) Spon1-positive areas were observed compared to Day 14. ( D ) The most Spon1-positive areas were observed compared to other groups. Scale bars = 100 μm; magnified image Bars = 50 μm. Ce: cementum, AB: alveolar bone. ( I ) The ratio of Spon1-positive area in the apical periodontal ligament calculated as: (Spon1-positive area/Area of interest) × 100%. Asterisks indicate significant differences among groups on the same postoperative day (**: p < 0.01), whereas daggers indicate significant differences between Day 14 and Day 28 within the same group (†: p < 0.05, ††: p < 0.01).

Article Snippet: In this study, we used F-spondin ( SPON1 ) mRNA (Hs01120488_m1) as a cementoblast differentiation marker.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Dentistry Journal

Article Title: Bone Morphogenetic Protein 7 Promotes the Differentiation of Periodontal Ligament Fibroblasts into F-Spondin-Expressing Cementoblast-like Cells During Root Canal Treatment—An In Vivo Rat Pulpectomy Model and In Vitro Human Fibroblast Study

doi: 10.3390/dj13110494

Figure Lengend Snippet: mRNA expression levels at each BMP-7 concentration assessed by qRT-PCR analysis. The expression levels of each mRNA were higher in the 100 ng/mL BMP-7-treated group compared to the control group. ( A ) SPON1 mRNA expression levels. ( B ) CEMP1 mRNA expression levels. ( C ) RUNX2 mRNA expression levels. ( D ) BGLAP mRNA expression levels. ( n = 4) Asterisks indicate significant differences among BMP-7 concentrations on the same day (*: p < 0.05, **: p < 0.01), whereas daggers indicate significant differences between Day 7 and Day 14 within the same BMP-7 concentration (†: p < 0.05, ††: p < 0.01).

Article Snippet: In this study, we used F-spondin ( SPON1 ) mRNA (Hs01120488_m1) as a cementoblast differentiation marker.

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Control

Journal: Dentistry Journal

Article Title: Bone Morphogenetic Protein 7 Promotes the Differentiation of Periodontal Ligament Fibroblasts into F-Spondin-Expressing Cementoblast-like Cells During Root Canal Treatment—An In Vivo Rat Pulpectomy Model and In Vitro Human Fibroblast Study

doi: 10.3390/dj13110494

Figure Lengend Snippet: Detection of proteins in HPLFs following treatment at each BMP-7 concentration by Western blot analysis. SPON1 and CEMP1 proteins were qualitatively detected, showing the strongest bands in the 100 ng/mL BMP-7-treated group, followed by the 200 ng/mL BMP-7-treated group, whereas minimal or no expression was observed in the control. Lane 1, 0 ng/mL BMP-7-treated group as a control; lane 2, 100 ng/mL BMP-7-treated group; lane 3, 200 ng/mL BMP-7-treated group. Representative qualitative results are shown.

Article Snippet: In this study, we used F-spondin ( SPON1 ) mRNA (Hs01120488_m1) as a cementoblast differentiation marker.

Techniques: Concentration Assay, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Venn diagram outlining filtering for membrane and secreted genes that are enriched in the FP. The list was curated based on LC-MS identification of proteins from hFpO conditioned media, enriched expression in primary human FP cells from scRNA seq datasets, and presence in membrane/secreted GO terms. (B) Graph displaying ranked fold-difference in expression of membrane/secreted FP genes ordered from most conserved to most divergent expression. Heatmap represents the proportion of studies that found the plotted genes to be essential from the DepMap Portal. (C) Violin plots of gene expression for the most (>10-fold) divergently expressed membrane/secreted in humans (left) compared to mouse (right) primary FP cells. SPON1, NTN1, and NRP2 are included as conserved controls between species.

Article Snippet: The following primary antibodies were used: anti-NTN1 antibody (1:1000 dilution, rabbit, Abcam, ab126729), anti-SHH antibody (1:1000 dilution, rabbit, Abcam, ab53281), anti-GAPDH (1:3000 dilution, mouse, Cell Signaling Technology, 2118S), anti-GALNT2 (1:1000 dilution, rabbit, Invitrogen, PA5-21541), and anti-SPON1 (1:1000 dilution, goat, Bio-Techne, AF3135).

Techniques: Membrane, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Comparison of gene expression levels of 14,852 orthologous genes (gray points) between mouse (E9-14) and human (CS12-19) FP cells. Black dotted line indicates 1:1 gene expression while the blue dotted lines indicate 2-and 10-fold human enrichment. Colored points indicate membrane and secreted FP-enriched genes and indicate degree of gene expression divergence between human and mouse. (B) Schematic of workflow for generating hFpO from pooled KO hiPS cells and subsequently assembling with day 22 hSpO to form hMA in an arrayed format. At 3 d.a.f., the assembloids were cleared and confocal imaged to visualize commissural axons guiding towards the hFpO. (C) Heatmap displaying min/max normalized, fold-change in ROBO3 fluorescence intensity of hSpO axons 150 μm pre- and 150 μm post-crossing into the hFpO (n = 2-3 assembloids per gene). Two-way ANOVA performed on measurements the most distal 50 μm of heatmap; F 31,1972 = 10.05, P < 0.0001, following Dunnett’s multiple comparison test comparing Control 1 to all other conditions: ****P <0.0001 for Control 1 vs GALNT2 , ****P <0.0001 for Control 1 vs PLD3 , ****P < 0.0001 for Control 1 vs NTN1 , and **P = 0.0025 for Control 1 vs SPON1 . (D) Representative z-projected immunocytochemistry images of control and pooled KO hMA expressing ROBO3 and FOXA2 to visualize ROBO3+ axons projecting across the hFpO boundary. Scale bar, 100 μm. (E) Plot profiles of normalized fold-change ROBO3 fluorescence intensity of KO pool hFpO hMA 150 μm pre- and 150 μm post-crossing into the hFpO from (C). Dotted lines indicate the non-targeting control groups while the solid lines indicate the target gene group. Data is presented as mean ± SEM. Gray shaded region indicates the 50 μm bin where ROBO3 expression as a measure of axon guidance was assessed for statistical significance in (C). (F) Schematic outlining the method for generating isogenic KO hiPS cell lines from the axon guidance screen candidates and subsequent isogenic validation of phenotypes identified in the axon guidance screen. (G) Representative z-projected immunocytochemistry images of control and isogenic KO hMA expressing ROBO3 and FOXA2 at 2 d.a.f. Scale bar, 200 μm. (H) Bar plot displaying the proportion of ROBO3 coverage relative to the area of the isogenic KO hFpO candidates from 1-3 d.a.f (shades of grey; n = 4-5 assembloids per condition). Data is presented as mean ± SEM. Two-way ANOVA, F 4,59 = 121.8, P < 0.0001, following Tukey’s multiple comparison test ****P < 0.0001.

Article Snippet: The following primary antibodies were used: anti-NTN1 antibody (1:1000 dilution, rabbit, Abcam, ab126729), anti-SHH antibody (1:1000 dilution, rabbit, Abcam, ab53281), anti-GAPDH (1:3000 dilution, mouse, Cell Signaling Technology, 2118S), anti-GALNT2 (1:1000 dilution, rabbit, Invitrogen, PA5-21541), and anti-SPON1 (1:1000 dilution, goat, Bio-Techne, AF3135).

Techniques: Comparison, Expressing, Membrane, Fluorescence, Control, Immunocytochemistry

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Inference of CRISPR edits (ICE; no border) and KO-score (border) of CRISPR edited hFpO at day 8 (dark blue) and day 11 (light blue) of differentiation. (B) Gene expression analysis (by RT-qPCR) of FOXA2 in control and CRISPR edited hFpO at day 8 and 11 of differentiation (pooled mRNA from 3 individual organoids). Color scale indicates the relative expression of FOXA2 normalized to GAPDH . (C) Representative brightfield images of control and CRISPR edited hiPS cells (top) and z-projection of immunocytochemical images hFpO (stained for FOXA2 and Reddot2 nuclear stain) at day 8 and 11 of differentiation (bottom). Scale bar 100 μm. (D) Quantification of FOXA2/RedDot2 fluorescence intensity of control and CRISPR edited hFpO at day 8 (light gray) and 11 (dark gray) of differentiation (n = 2-3 organoids per condition). Data is normalized to negative control (hSpO). Data is presented as mean ± SEM. (E) ICE (no border) and KO-score (border) of isogenic CRISPR edited hiPS cell lines for GALNT2, NTN1 , and SPON1 knockout. (F) Western blot validation of hSpO and hFpO cell extracts and conditioned media collected from control and isogenic KO for GALNT2 (top), NTN1 (middle), and SPON1 (bottom). GAPDH immunoblotting was used as the sample loading control. (G) Gene expression analysis (by RT-qPCR) of FOXA2, PAX6 , and NKX2 . 2 in control and isogenic CRISPR edited hFpO at day 8 of differentiation. Each dot represents a separate differentiation experiment normalized to GAPDH expression. Data is presented as mean ± SEM. Two-tailed Mann-Whitney test was used. For FOXA2 : n = 4 control and 4 GALNT2 KO from 1 hiPS cell line; *P = 0.0286. For NKX2-2 : n = 4 control and 4 GALNT2 KO from 1 hiPS cell line; *P = 0.0286. For PAX6 : n = 4 control and 4 GALNT2 KO from 1 hiPS cell line; P = 0.0571.

Article Snippet: The following primary antibodies were used: anti-NTN1 antibody (1:1000 dilution, rabbit, Abcam, ab126729), anti-SHH antibody (1:1000 dilution, rabbit, Abcam, ab53281), anti-GAPDH (1:3000 dilution, mouse, Cell Signaling Technology, 2118S), anti-GALNT2 (1:1000 dilution, rabbit, Invitrogen, PA5-21541), and anti-SPON1 (1:1000 dilution, goat, Bio-Techne, AF3135).

Techniques: CRISPR, Expressing, Quantitative RT-PCR, Control, Staining, Fluorescence, Negative Control, Knock-Out, Western Blot, Two Tailed Test, MANN-WHITNEY