rabbit anti human runx2  (Danaher Inc)

Journal: JBMR Plus

Article Title: Proteomic analysis and effects on osteogenic differentiation of exosomes from patients with ossification of the spinal ligament

doi: 10.1093/jbmrpl/ziaf021

Figure Lengend Snippet: (A) Western blots revealing Runx2 and Wnt3a expression at wk 2 and 3 following culture in osteogenic differentiation medium using exosome-penetrating (1.2 μm) and exosome-non-penetrating (0.03 μm) filters. (B) Expression levels of Runx2 and Wnt3a of OLF-derived and non-OLF-derived (control) cells co-cultured with 0.03- and 1.2-μm filters. ( n = 3 each). ** p <.01.

Article Snippet: The membranes were washed twice in PBS solution containing 0.05% Tween 20, then blocked by a mixture of 5% skimmed milk in PBS for 1 h at room temperature, and finally incubated overnight with one of the following antibodies at 4 °C: rabbit anti-Runx2 polyclonal Ab (dilution, 1:250, Proteintech) and rabbit anti-Wnt3a polyclonal Ab (dilution, 1:250, Proteintech) to assess the osteogenic differentiation, and rabbit anti-COLIVA1 polyclonal Ab (dilution, 1:250, GeneTex) rabbit anti-FMNL3 polyclonal Ab (dilution, 1:250, Proteintech), rabbit anti-mTOR polyclonal Ab (dilution, 1:250, Proteintech), or rabbit anti-PIP4K2B polyclonal Ab (dilution, 1:250, Proteintech) to confirm the differential expression of candidate proteins.

Techniques: Western Blot, Expressing, Derivative Assay, Control, Cell Culture

Journal: Communications Biology

Article Title: Dental pulp stem cells-derived cannabidiol-treated organoid-like microspheroids show robust osteogenic potential via upregulation of WNT6

doi: 10.1038/s42003-024-06655-y

Figure Lengend Snippet: ALP, BMP2, RUNX2, OCN, and OPN mRNA expression at day 7 ( A ) and at day 14 ( B ). C Representative western blots of ALP, BMP2, COL-1, and RUNX2 expression at day 14 and quantification ( D ). E The images of the frozen sections obtained from microspheroids cultured at 14 days under osteogenic medium with or without CBD stained with immunofluorescence and F quantification. Data were presented as the mean ± SD ( n = 3). Significant difference compared different groups, * P < 0.05, ** P < 0.01, and *** P < 0.001; compared between DPSC and CBD+DPSC groups in blue; compared between DPSC and spheroids groups in red; compared between spheroids and CBD+spheroids groups in green; compared between CBD+DPSC and CBD+spheroids groups in purple.

Article Snippet: The membrane was then incubated at 4 °C overnight with primary antibodies, which included rabbit anti-ALP (Affinity, China), rabbit anti-BMP2 (Abcam, England), rabbit anti-OCN (Abcam, England), rabbit anti-human Collagen I (COL-I, Abcam, England), rabbit anti-human RUNX2 (Abcam, England), rabbit anti-human WNT6 (Bioss, China), and rabbit anti-human β-catenin (Bioss, China).

Techniques: Expressing, Western Blot, Cell Culture, Staining, Immunofluorescence

Journal: Communications Biology

Article Title: Dental pulp stem cells-derived cannabidiol-treated organoid-like microspheroids show robust osteogenic potential via upregulation of WNT6

doi: 10.1038/s42003-024-06655-y

Figure Lengend Snippet: A Venn diagram showing the differentially expressed genes (DGEs) between four groups. B KEGG analysis of common DEGs. C Heatmap displaying the Wnt/β-catenin pathway-related DGEs between DPSC and microspheroids with or without CBD. D Quantification of differentially expressed WNT6 analyzed based on FPKM. E mRNA expression of WNT6. F Western blot analysis of WNT6, β-catenin, and quantification ( G ). H Western blot analysis of β-catenin, OCN and RUNX2 protein expression of microspheroids upon DKK1 treatment for 48 h and quantification ( I ). Data were presented as the mean ± SD ( n = 3). Significant difference compared different groups, * P < 0.05, ** P < 0.01, and *** P < 0.001; compared between DPSC and CBD+DPSC groups in blue; compared between DPSC and spheroids groups in red; compared between spheroids and CBD+spheroids groups in green; compared between CBD+DPSC and CBD+spheroids groups in purple.

Article Snippet: The membrane was then incubated at 4 °C overnight with primary antibodies, which included rabbit anti-ALP (Affinity, China), rabbit anti-BMP2 (Abcam, England), rabbit anti-OCN (Abcam, England), rabbit anti-human Collagen I (COL-I, Abcam, England), rabbit anti-human RUNX2 (Abcam, England), rabbit anti-human WNT6 (Bioss, China), and rabbit anti-human β-catenin (Bioss, China).

Techniques: Expressing, Western Blot

Journal: Communications Biology

Article Title: Dental pulp stem cells-derived cannabidiol-treated organoid-like microspheroids show robust osteogenic potential via upregulation of WNT6

doi: 10.1038/s42003-024-06655-y

Figure Lengend Snippet: Primers used in RT-qPCR to examine gene expression

Article Snippet: The membrane was then incubated at 4 °C overnight with primary antibodies, which included rabbit anti-ALP (Affinity, China), rabbit anti-BMP2 (Abcam, England), rabbit anti-OCN (Abcam, England), rabbit anti-human Collagen I (COL-I, Abcam, England), rabbit anti-human RUNX2 (Abcam, England), rabbit anti-human WNT6 (Bioss, China), and rabbit anti-human β-catenin (Bioss, China).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

doi: 10.1101/2023.07.28.550935

Figure Lengend Snippet: A) Overview of the human RUNX2 coding sequence comprising 8 exons. gRNAs and their corresponding expected protein structure. The gRNA targeting exon 2 (orange) disrupts the DNA binding domain, gRNA targeting exon 5 (violet) disrupts nuclear translocation, gRNAs targeting exon 6 (blue) disrupt the transcriptional activation domain and gRNAs targeting exon 8 (green) disrupt the nuclear matrix targeting signal and repress the protein function. B) Intracellular flow cytometry for RUNX2 detection in MSOD-B and RUNX2-edited clones. A clear protein reduction could be observed in the 6.1_1 and 6.1_23 clones. C) Western blot analysis of RUNX2 in cultured MSOD-B and RUNX2-edited cells. The genetic editing of RUNX2 is confirmed by the detection of the truncated proteins. Actin is used as a control to normalize the protein content. D) Histological analysis of in vitro constructs using Safranin O (top) and Masson’s trichrome (bottom) stains, indicating the presence of cartilage matrices (Scale bars = 200 μm). E) Quantitative assessment of the total GAG content in corresponding in vitro generated tissues. F) Immunofluorescence images of MSOD-B and MSOD-BΔR1 tissues. Displayed images consist of 3D-stacks from 80-100 μm thick sections, stained for DAPI (blue), Collagen Type II (COL2, yellow) and Collagen Type X (COLX, red). A clear reduction in the COLX signal could be observed in the MSOD-BΔR1 tissues, indicating impaired hypertrophy. G) Isosurface-based quantification of the COL2 immunofluorescent signal using the IMARIS software. No significant difference between groups confirm the retention of cartilage formation in RUNX2-edited constructs. H) Isosurface-based quantification of the COLX immunofluorescent signal using the IMARIS software, confirming the disruption of hypertrophy in the MSOD-BΔR1 constructs. Unpaired t-test, n=3, *p < 0.05, n.s. = not significant.

Article Snippet: Following a blocking step in 10% normal donkey serum (Sigma), cells were stained with a primary antibody against RUNX2 (Rabbit anti-human, Thermo-Fischer PA5-82787) for one hour at room temperature.

Techniques: Sequencing, Binding Assay, Translocation Assay, Activation Assay, Flow Cytometry, Clone Assay, Western Blot, Cell Culture, In Vitro, Construct, Generated, Immunofluorescence, Staining, Software