Journal: PLoS Genetics

Article Title: The DNA Helicase Recql4 Is Required for Normal Osteoblast Expansion and Osteosarcoma Formation

doi: 10.1371/journal.pgen.1005160

Figure Lengend Snippet: (A) Kaplan-Meier survival plots of Osx -Cre Recql4 (left) and Osx -Cre p53 fl/fl Recql4 (right) mice. P value calculated by Log-Rank statistical test. (B) H&E stained sections of primary OS tumors from Osx -Cre p53 fl/fl Recql4 animals of indicated genotype. (C) Representative reconstructed μCT images of primary OS tumors from Osx -Cre p53 fl/fl Recql4 fl/+ and Recql4 fl/fl mice. (D) Flow cytometry percentages of tumor cells stained with CD51, Sca1 and PDGFRα. n = 3 for Recql4 +/+ , n = 8 for Recql4 fl/+ , n = 7 for Recql4 fl/fl ; Data presented as mean±SEM. (E) Representative photos of Alizarin Red-stained tumor cells that were subjected to osteogenic differentiation conditions. (F) qPCR profiling of Osx -Cre p53 fl/fl Recql4 +/+ , Recql4 fl/+ and Recql4 fl/fl tumors for the indicated genes. n = 3–4; Data presented as mean±SEM. (G) Assessment of genomic excision of Recql4 in tumor-derived cell lines. 40ng of genomic DNA was used for PCR and subjected to gel electrophoresis.

Article Snippet: The Kusa4b10, long-bone primary osteoblastic cells and mouse OS cell lines (no authentication performed) were cultured in αMEM (Lonza), 10% non heat inactivated FBS (SAFC Biosciences) and 1% Penicillin/Streptamycin/Glutamine (Life Technologies).

Techniques: Staining, Flow Cytometry, Derivative Assay, Nucleic Acid Electrophoresis

Journal: Marine Drugs

Article Title: Physicochemical and Biocompatibility Properties of Type I Collagen from the Skin of Nile Tilapia ( Oreochromis niloticus ) for Biomedical Applications

doi: 10.3390/md17030137

Figure Lengend Snippet: The effect of ASC, PSC, and porcine collagen (PC) on cell proliferation. ( A ): MC3T3E1, ( B ): L929, ( C ): HUVEC. Values with * show significant differences ( p < 0.05) between groups, as determined by one-way ANOVA.

Article Snippet: MC3T3E1 and L929 cells were obtained from a cell bank (Procell Life Science & Technology Co., Ltd., Wuhan, China) and HUVEC were received from an institute of zoology (Chinese academy of sciences, Beijing, China).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: The Study of Nanosized Silicate-Substituted Hydroxyapatites Co-Doped with Sr 2+ and Zn 2+ Ions Related to Their Influence on Biological Activities

doi: 10.3390/cimb44120425

Figure Lengend Snippet: Influence of Zn 2+ and Sr 2+ ions co-doped silicate-substituted hydroxyapatite nanoparticles on the viability of ( A ) TC28A2 and ( B ) 7F2 cell lines.

Article Snippet: The TC28A2 human chondrocyte cell line and 7F2 mouse osteoblast cell line were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine (Biowest, Nuaillé, France) and supplemented with 10% Fetal Bovine Serum (FBS) South America Heat Inactivated (Biowest), 200 U/mL penicillin and 200 μg/mL streptomycin.

Techniques:

Journal: PeerJ

Article Title: miR-452-3p inhibited osteoblast differentiation by targeting Smad4

doi: 10.7717/peerj.12228

Figure Lengend Snippet: (A) The cell viability of MC3T3-E1 after 200 ng/mL BMP2 treatment for 0, 12, 24, 36, and 48 h was assessed using CCK-8 assay. (B) The number of calcium nodules was counted in 200 ng/mL BMP2-treated MC3T3-E1 cells by alizarin red staining, compared with untreated MC3T3-E1 group. (C) On days 0, 7, and 14, the mRNA levels of the osteoblast marker genes OPN, Col1A1, and Runx2 were assessed using by RT-PCR in induced MC3T3-E1 cells. (D) Alkaline phosphatase (ALP) activity in BMP2-treated MC3T3-E1 cells on days 0, 7 and 14 was measured using a commercial assay kit. (E) RT-PCR analysis of the expression of miR-452-3p in induced MC3T3-E1 cells. N = 3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The mouse osteoblast cell line (MC3T3-E1) was purchased from ScienCell (Carlsbad, CA, USA) and cultured in alpha-minimum essential medium (α-MEM; SH30265.01B; Hyclone, South Logan, UT, USA) containing 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 10% foetal bovine serum (FBS).

Techniques: CCK-8 Assay, Staining, Marker, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Expressing

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Chromatin accessibility landscapes of Fam20c -deficient osteoblasts. A Landscape of chromatin accessibility from ATAC-seq analysis in OB Fam20c f/f and OB Fam20c KO . B Volcano plot of all peaks. Differentially accessible regions up-regulated in OB Fam20c KO compared with OB Fam20c f/f are indicated as red dots, the differentially accessible regions down-regulated are indicated as blue dots, and the grey dots indicate peaks with no significant difference. The x-axis represents log2 (Fold Change), and the y-axis indicates -log10 ( P value). C Bar chart showing the distribution of up- and down-regulated differentially accessible regions in OB Fam20c KO relative to gene features. The X-axis represents the proportion of peaks in each functional area, and the Y-axis represents different groups. The genome-wide functional regions were divided into promoter, 5’UTR, 3’UTR, exon, intron, and intergenic regions. D Up regulated motifs enriched in OB Fam20c KO enhancers compared with OB Fam20c f/f (top 10 motifs displayed). E Down regulated motifs enriched in OB Fam20c KO enhancers compared with OB Fam20c f/f (top 10 motifs displayed)

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Functional Assay, Genome Wide

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Motif enrichment results down regulated in OB Fam20c KO (top 20 displayed)

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Significance Assay

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Analysis of ATAC-seq and intersection with RNA-seq in Fam20c deficient osteoblast. A Venn diagram depicts the intersection of genes with DAR-associated up-regulated and up-regulated DEGs in OB Fam20c KO . B Venn diagram depicts the intersection of genes with DAR-associated down-regulated and down-regulated DEGs in OB Fam20c KO . C Biological Processes of Gene Ontology (GO) enrichment analysis. The colors of circle dots illustrate the P -values identified for each GO term (low: red, high: blue), with lower values for more significant enrichment. The size of dots indicates the number of the differentially expressed genes and the larger dots represent a larger gene number. D Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The colors of circle dots illustrate the P -values identified for each GO term (low: red, high: blue), with lower values for more significant enrichment. The size of dots indicates the number of the differentially expressed genes and the larger dots represent a larger gene number

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: RNA Sequencing

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Analysis of Proteomics and Phosphoproteomics in Fam20c deficient osteoblast. A Clustered heatmap of top 40 (20 up-regulated and 20 down-regulated) differentially expressed proteins between OB Fam20c f/f and OB Fam20c KO in proteomics. B Clustered heatmap of top 40 (20 up-regulated and 20 down-regulated) differentially expressed proteins between OB Fam20c f/f and OB Fam20c KO in phosphoproteomics. C The enrichment of differentially expressed proteins between OB Fam20c f/f and OB Fam20c KO were analyzed via gene set enrichment analysis (GSEA). D The enrichment of differentially expressed phosphorylated proteins between OB Fam20c f/f and OB Fam20c KO were analyzed via gene set enrichment analysis (GSEA). E Venn diagram depicts the intersection of proteins with differentially expressed proteins and differentially expressed proteins. The upper diagram is the up-regulated part, below depicts down-regulated part. F Volcano plot of differentially expressed proteins based on proteomics data with 9 intersected proteins labeled. G Volcano plot of differentially expressed phosphorylated proteins based on phosphorylated proteomics data with 9 intersected proteins labeled

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Phospho-proteomics, Labeling

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Fam20c affects the Calpastatin/Calpain proteolysis system. A Expression (qPCR) of Calpastatin , Calpain 1, and Calpain 2 in OB Fam20c f/f and OB Fam20c KO . * P < 0.05, *** P < 0.001. B Western blot analysis of Calpastatin, Calpain 1, and Calpain 2 expression in OB Fam20c f/f and OB Fam20c KO . GAPDH served as an internal control. C Phos-Tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (P-tag) demonstrates Calpastatin phosphorylation levels in OB Fam20c f/f and OB Fam20c KO . GAPDH served as an internal control. D Casein zymography profiles of Calpain 1 and Calpain 2 in OB Fam20c f/f and OB Fam20c KO . E Immunofluorescence staining for Calpastatin, Calpain 1, and Calpain 2 in OB Fam20c f/f and OB Fam20c KO . Scale bar = 20 µm. F Gene expression of Calpastatin , Calpain 1, and Calpain 2 in Ctrl and cKO mice femurs. * P < 0.05, ** P < 0.01. G Immunohistochemical staining for Calpastatin in 4-week-old Ctrl and cKO mice femurs. (A1) and (B1) were free bone trabeculae in the bone marrow. (A2) and (B2) were the site of bone formation in the calcified cartilage. (A3) and (B3) were the junction of the cartilage proliferation area and hypertrophic area. Red box represented local magnification, scale bar = 50 μm. (a1), (a2), (a3), (b1), (b2), (b3) were the corresponding magnification regions, scale bar = 20 μm. H Immunohistochemical staining for Calpain 1 and Calpain 2 in 4-week-old Ctrl and cKO mice femurs. Scale bar = 50 μm

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Expressing, Western Blot, Control, Polyacrylamide Gel Electrophoresis, SDS Page, Phospho-proteomics, Zymography, Immunofluorescence, Staining, Gene Expression, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Migration and F-actin behavior in response to knockout of Fam20c in osteoblast. A Wounding healing assays of OB Fam20c f/f and OB Fam20c KO , images were acquired 0 h and 24 h after the scratch. Scale bar = 100 μm. B Bar graph depicting cell migration rate (%) in a scratch wound-healing assay in OB Fam20c f/f and OB Fam20c KO . ** P < 0.01, t test. C OB Fam20c f/f and OB Fam20c KO were stained by Phalloidin and DAPI to visualize F-actin and nucleus, respectively. OB Fam20c KO showed a decreased amount of F-actin formation as compared with OB Fam20c f/f . White arrowheads indicate heavily polymerized F-actin. Scale bar = 100 µm. D Gene expression of Rac1 , RhoA , and Cdc42 in OB Fam20c f/f and OB Fam20c KO . * P < 0.05. E Western blot analysis of Cdc42 and RhoA expression in OB Fam20c f/f and OB Fam20c KO . GAPDH served as an internal control

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Migration, Knock-Out, Wound Healing Assay, Staining, Gene Expression, Western Blot, Expressing, Control

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Calpastatin/Calpain proteolysis system influences cell migration velocity and F-actin behavior in Fam20c -deficient cells. A Gene expression of Calpastatin , Calpain 1, and Calpain 2 in OB Fam20c KO and OB Fam20c KO treated with CaCl 2 . * P < 0.05, ** P < 0.01, **** P < 0.0001. B Western blot analysis of Calpastatin, Calpain 1, and Calpain 2 expression in OB Fam20c KO and OB Fam20c KO treated with CaCl 2 . GAPDH served as an internal control. C Gene expression of Calpastatin , Calpain 1 , and Calpain 2 in OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with different concentrations of CaCl 2 . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS: no significance. D Phos-Tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (P-tag) demonstrates effects of CaCl 2 on Calpastatin phosphorylation levels in OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with CaCl 2 . Tubulin served as an internal control. E Casein zymography profiles of Calpain 1 and Calpain 2 in OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with CaCl 2 . F Wounding healing assays of OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with CaCl 2 , images were acquired 0 h and 24 h after the scratch. Scale bar = 100 μm. G Gene expression of Rac1 , RhoA , and Cdc42 in OB Fam20c KO and OB Fam20c KO treated with CaCl 2 . * P < 0.05, ** P < 0.01. H Western blot analysis of Cdc42 expression in OB Fam20c f/f , OB Fam20c KO and OB Fam20c KO treated with CaCl 2 . GAPDH served as an internal control

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Migration, Gene Expression, Western Blot, Expressing, Control, Polyacrylamide Gel Electrophoresis, SDS Page, Phospho-proteomics, Zymography

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Fam20c affects the Wnt signaling pathway. A Gene expression of β-catenin , Tcf4, and GSK-3β in OB Fam20c f/f and OB Fam20c KO . * P < 0.05, ** P < 0.01, *** P < 0.001. B Western blot analysis of β-catenin, Tcf4, and GSK-3β expression in OB Fam20c f/f and OB Fam20c KO . GAPDH served as an internal control. C Western blot analysis of β-catenin, Tcf4, and GSK-3β of nuclear and cytoplasm expression in OB Fam20c f/f and OB Fam20c KO . GAPDH served as an internal control. D Immunofluorescence staining for β-catenin in OB Fam20c f/f and OB Fam20c KO . Scale bar = 100 µm. E Immunofluorescence staining for Tcf4 in OB Fam20c f/f and OB Fam20c KO . Scale bar = 100 µm. F Immunofluorescence staining for GSK-3β in OB Fam20c f/f and OB Fam20c KO . Scale bar = 100 µm. G RNA-seq data in OB Fam20c f/f and OB Fam20c KO . Fold represents log2fold change. H Immunohistochemical staining for GSK-3β in 4-week-old Ctrl and cKO mice femurs. The black arrows showed the area where cartilage calcifies to form bone trabecular, and the red arrows indicated a hypertrophic layer of cartilage. Scale bar = 50 μm. I Immunohistochemical staining for Tcf4 in 4-week-old Ctrl and cKO mice femurs. Scale bar = 50 μm. J Immunohistochemical staining for β-catenin in 4-week-old Ctrl and cKO mice femurs. (A1) and (B1) were proliferating layers of articular cartilage of the femur. (A2) and (B2) were the site of the hypertrophic cartilage layer on the surface of a joint. Red box represented local magnification, (A1), (A2), (B1), (B2), scale bar = 50 μm. (a1), (a2), (b1), (b2), were the corresponding magnification regions, scale bar = 20 μm. K Western blot analysis of β-catenin, Tcf4, and GSK-3β expression in OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with CaCl 2 . GAPDH served as an internal control. L Immunofluorescence staining for β-catenin in OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with CaCl 2 . Scale bar = 100 µm. M Immunofluorescence staining for Tcf4 in OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with CaCl 2 . Scale bar = 100 µm. N Immunofluorescence staining for GSK-3β in OB Fam20c f/f , OB Fam20c KO , and OB Fam20c KO treated with CaCl 2 . Scale bar = 100 µm

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Gene Expression, Western Blot, Expressing, Control, Immunofluorescence, Staining, RNA Sequencing, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

doi: 10.1186/s12967-023-04275-4

Figure Lengend Snippet: Motif enrichment results up regulated in OB Fam20c KO (top 20 displayed)

Article Snippet: Immortalized mouse Fam20c f/f osteoblast cells (referred as “OB Fam20c f/f ”) were purchased from Applied Biological Materials Inc. (Lot# T0038).

Techniques: Significance Assay