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: Oncotarget

Article Title: Identification of novel genes in aging osteoblasts using next-generation sequencing and bioinformatics

doi: 10.18632/oncotarget.22748

Figure Lengend Snippet: The morphological changes ( A ) and β-galactosidase staining ( B ) of human primary osteoblasts of second, fourth, and seventh passages were identified. The morphologies of young osteoblasts were thin and spindle-shaped. Increased number of cells with flattened and irregularly-shaped morphologies and increased intracellular debris were observed at later passages (arrow). The cells with blue staining indicated β-galactosidase-positive cells (arrow), representing senescent cells. The cells were observed under light microscope at 10X field.

Article Snippet: The first passage of primary osteoblasts, delineated as P1, was defined as cells harvested from directly-thawed vials purchased from Lonza under stable growth conditions.

Techniques: Staining, Light Microscopy

Journal: Oncotarget

Article Title: Identification of novel genes in aging osteoblasts using next-generation sequencing and bioinformatics

doi: 10.18632/oncotarget.22748

Figure Lengend Snippet: ( A ) Next generation sequencing (NGS) analysis identified 504 up-regulated genes and 385 down-regulated genes in human primary osteoblasts of eighth passage (P8), compared to first passage (P1) (> 2.0-fold change), where 399 up-regulated genes and 355 down-regulated genes were mapped using Ingenuity Pathway Analysis (IPA) database. In addition, 10 up-regulated microRNAs and 19 down-regulated microRNAs were selected (> 2.0-fold change and threshold of reads per million (RPM) >10), which predicted 381 and 606 putative targets by miRmap (score≥ 99.0), respectively. Among all identified putative targets, 378 targets predicted by 10 up-regulated microRNAs and 599 targets predicted by 19 down-regulated microRNAs were mapped in IPA database. These selected targets and differentially expressed genes were matched by the “Compare Dataset” tool in the IPA, and revealed 22 potential microRNA-mRNA interactions. * One of the mapped genes was not identified in our NGS dataset. ( B ) The heatmap analysis of differentially expressed microRNAs from P1 and P8 osteoblasts with z-score values were shown.

Article Snippet: The first passage of primary osteoblasts, delineated as P1, was defined as cells harvested from directly-thawed vials purchased from Lonza under stable growth conditions.

Techniques: Next-Generation Sequencing

Journal: Oncotarget

Article Title: Identification of novel genes in aging osteoblasts using next-generation sequencing and bioinformatics

doi: 10.18632/oncotarget.22748

Figure Lengend Snippet: ( A ) The 22 candidate genes identified from human osteoblasts were analyzed by IPA for network analysis. The network analysis revealed 10 out of 22 genes involved in a network associated with cancer, endocrine system disorders, organismal injury and abnormalities, and three of the four putative targets ( KLF7 , SOX11 and SVIP ) by miR-204-5p and miR-335-3p predictions were involved. Genes colored in green indicated down-regulated expressions, and genes in red indicated up-regulated expressions in our dataset. ( B ) miR-204-5p was analyzed by IPA as a potential upstream regulator in our dataset. Using the overlay tool in IPA, molecules involved in the canonical osteoarthritis pathway, Wnt/β-catenin pathway, and differentiation of osteoblasts were marked in purple, where SOX11 was the molecule simultaneously involved.

Article Snippet: The first passage of primary osteoblasts, delineated as P1, was defined as cells harvested from directly-thawed vials purchased from Lonza under stable growth conditions.

Techniques:

Journal: Oncotarget

Article Title: Identification of novel genes in aging osteoblasts using next-generation sequencing and bioinformatics

doi: 10.18632/oncotarget.22748

Figure Lengend Snippet: The differentially expressed genes identified in our NGS dataset were analyzed by IPA and categorized into 25 networks, where skeletal diseases and functions annotation, including bone mineral density, differentiation of osteoblasts, osteoarthritis and damage of cartilage tissue were selected to identify related genes. Merged network analysis showed SOX11 was interconnected between the networks of osteoblast differentiation and bone mineral density, and was predicted to inhibit EGR1 and activate PRDM1 , two molecules in the bone mineral density network.

Article Snippet: The first passage of primary osteoblasts, delineated as P1, was defined as cells harvested from directly-thawed vials purchased from Lonza under stable growth conditions.

Techniques:

Journal: Oncotarget

Article Title: Identification of novel genes in aging osteoblasts using next-generation sequencing and bioinformatics

doi: 10.18632/oncotarget.22748

Figure Lengend Snippet: The proposed novel molecular mechanisms of gene regulations involved in aging osteoblasts

Article Snippet: The first passage of primary osteoblasts, delineated as P1, was defined as cells harvested from directly-thawed vials purchased from Lonza under stable growth conditions.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Immune regulation by glucocorticoids can be linked to cell type–dependent transcriptional responses

doi: 10.1084/jem.20180595

Figure Lengend Snippet: The direction and magnitude of transcriptional regulation by glucocorticoids are cell type dependent. Four primary human hematopoietic cell types and five primary human nonhematopoietic cell types were studied. For each cell type, cells from four unrelated healthy donors were independently cultured and treated with methylprednisolone (22.7 µM) or vehicle (0.08% ethanol). Total RNA was purified 2 and 6 h after in vitro treatment and RNA-seq was performed. Differential expression was assessed by comparing data from methylprednisolone-treated versus vehicle-treated cells in the four biological replicates. The statistical significance of differential expression was calculated with a Wald test, after accounting for dispersion, library size, and read count. The resulting P values for differential expression were adjusted for multiple testing by the method of . (a) The left panel displays the transcriptional response to glucocorticoids in hematopoietic cells versus nonhematopoietic cells for each of 56,870 genes. The log2 fold change compares methylprednisolone-treated versus vehicle-treated cells after 6 h of in vitro treatment. Each dot represents one gene. The x-axis variable is the mean log2 fold change in the five nonhematopoietic cells (endothelial cells, fibroblasts, myoblasts, osteoblasts, and preadipocytes), and the y-axis variable is the mean log2 fold change (FC) in the four hematopoietic cells (B cells, CD4 + T cells, monocytes, and neutrophils). The four tails of the distribution are color-coded and represent genes with evidence of transcriptional response to glucocorticoid (defined here as a mean log2 fold change ≥ 0.5 or ≤ −0.5) in one group of cells but not in the other. The right panel displays the baseline expression levels in hematopoietic versus nonhematopoietic cells for the genes with strongest evidence of a transcriptional response to glucocorticoid in one group of cells but not in the other (genes at the four tails of the distribution, as defined above). The values displayed are the mean log2 normalized read count at baseline in nonhematopoietic cells (x axis) versus hematopoietic cells (y axis). (b) Transcriptional response of TRIM22 to in vitro glucocorticoid treatment in nine primary human cell types. (c) Transcriptional response of ITGA5 to in vitro glucocorticoid treatment in nine primary human cell types. In b and c, the values displayed are the normalized read counts in vehicle-treated cells (VH; average of 2 and 6 h) and in glucocorticoid-treated cells (GC; 2 or 6 h). Each dot represents one biological replicate (one donor). Multiple-testing-adjusted P values ( q ) are from comparisons of glucocorticoid-treated versus vehicle-treated cells at each of the two time points. ns, not significant ( q > 0.05).

Article Snippet: Human primary osteoblasts from adult (Lonza; cat. no. CC-2538, lot no. 0000435102) or child (Lonza; cat. no. CC-2538, lot nos.

Techniques: Cell Culture, Purification, In Vitro, RNA Sequencing, Quantitative Proteomics, Dispersion, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Optimal intensity of ESW (10 kV for 500 impulses) accelerated osteoblast adhesion. Data are presented as the mean ± S.D. (error bars) in triplicate independent experiments (n = 3). The data show that at the times of 2, 4, 6, 8, and 10 h after ESW treatment, the number of adhesive osteoblasts was significant higher than the number without ESW treatment. p < 0.01 as compared with the control group at the same period. When the siItgb1 was added prior to ESW treatment, the promotion of adhesion of osteoblasts by ESW was inhibited. p < 0.01 as compared with the ESW group at the same period. p > 0.05 as compared with the control group at the same period. It was observed that siItga5 also inhibited the ESW-induced adhesion although not as significantly as did siItgb1. The promotion of adhesion induced by ESW was abrogated, whereas integrin α5 and β1 subunits were silenced. p < 0.01 as compared with the ESW group at the same period.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Adhesive, Control

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: ESW promoted migration of osteoblasts as shown in transwell tests and wound healing assays. The promotion could be inhibited by both siItgb1 and siItga5. Primary cultured osteoblasts were divided into six groups randomly; those were cells with (SW) (B1–B3) or without 10 kV for 500 impulses of ESWT (Control) (A1–A3) and with negative siRNA control (SW + siRNA control) (C1–C3), siItga5 (SW + siItga5) (E1–E3), siItgb1 (SW + siItgb1) (D1–D3), or both siItga5 and siItgb1 (F1–F3) for 6 h prior to ESWT. Results from the wound healing assays (G) and transwell tests (H) were consistent. Data are presented as the mean ± S.D. (error bars) (n = 6). a, p < 0.01; b, p < 0.05; c, p > 0.05 as compared with the control group. #, p > 0.05 as compared with the ESWT group. Scale bars, 100 μm.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Migration, Cell Culture, Control

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: A–D, ESW-induced elevations of mRNA level of α5 and β1 integrin of osteoblasts, peaking at 1 h (B). The specific inhibitors for signal transduction pathways had no influence on the integrin expression (C). A and C, representative electrophoretic images. The osteoblasts were harvested to extract total RNA 0.5, 1, 2, 4, 8, and 12 h after 500 impulses of 10-kV shock wave treatment. The cells without ESWT were run as control groups. After standardization of housekeeping gene expression, equal amounts of cDNA from each sample were subjected to 36 cycles to amplify Itga5 and Itgb1 mRNA expression. The values of the control group were normalized to 100%. a, p > 0.05; b, p < 0.01; c, p < 0.05 as compared with the control group at certain time periods. In addition, several signal transduction pathway inhibitors were added to the samples for 1 h prior to ESWT. 2 h after ESWT, the samples were collected to extract RNA and to analyze whether the Itga5 and Itgb1 mRNA were influenced by signal pathway inhibitors listed above. Our data showed that no influence on the expression of Itga5 or Itgb1 mRNA was observed under the conditions with or without inhibitors (p > 0.05). Error bars, S.D.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Transduction, Expressing, Control, Gene Expression

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: ESW enhanced integrin α5 and β1 subunit protein production in 2 h according to the data from flow cytometry analysis and Western blotting. For flow cytometry, osteoblasts from experimental groups and the control group were stained with PE-conjugated anti-rat Itga5 or Itgb1 antibody under the guidance of the manufacturer. It is shown that both Itga5 and Itgb1 proteins increased significantly in 2 h in the experimental group (A). *, p = 0.021; #, p < 0.01 as compared with the blank control group. Samples with or without ESWT and those associated with siItga5 and/or siItgb1 prior to ESW were subjected to radioimmune precipitation assay lysis. Western blotting was applied to analyze whether Itga5 and Itgb1 expression levels changed in the protein extractions. The same results are shown as those indicated by flow cytometry (B and C). In addition, the data from the ESW plus siRNA groups indicated that the siRNA reagents for both Itga5 and Itgb1 were effective (B and C). a, p < 0.05; b, p < 0.01 as compared with the blank control group. #, p < 0.01 as compared with ESW group. Results are presented with mean values ± S.E. (error bars) calculated from four paired triplicate experiments.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Flow Cytometry, Western Blot, Control, Staining, Lysis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: ESW activated ERK phosphorylation after treatment. ERK1/2 phosphorylation was increased in 2 h (A and C). The higher production of phosphorylated ERK1/2 persisted for 4 h (A and C). Osteoblasts were serum-deprived for 12 h before treatment with 10 kV for 500 impulses in the absence or presence of U0126 and siRNAs for the indicated time. Western blotting analysis was performed with antibodies against ERK and its phosphorylated forms (p-ERK) (B). C and D, summary of the results (mean ± S.E. (error bars), n = 4, triplicate in each experiment). a, p > 0.05 as compared with the control at the same period. b, p < 0.01 as compared with the control at the same period. c, p > 0.05 as compared with the control group. d, p < 0.01 as compared with the ESW group.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Evaluations of phosphorylation levels of focal adhesion kinase surrounding Tyr-397, Tyr-576/577, and Tyr-925. After experimental groups were subjected to direct exposure to 10 kV for 500 impulses of ESWT, we collected the extracts at 0.5, 1, 2, 4, or 8 h. Samples without ESWT were set as the control group. Then the extracts were quantified in triplicate using Western blotting and normalized by β-actin expression (A). A marked elevation of FAK phosphorylation (p-FAK) at Tyr-397 peaking at 4 h was observed, and we also observed a slight increase of FAK phosphorylation at Tyr-925 in 2 h after ESWT. ESW had no influence on the expression of FAK phosphorylation at Tyr-576/577 (Fig. 5A). A representative electrophoretic image of the study on FAK phosphorylation at Tyr-397 influenced by siRNAs of integrins is also depicted (B). 4 h after the optimal dose of ESWT, a decline in the expression of phosphorylated FAK at Tyr-397 was observed in the group with siRNA pretreatment. However, total protein expression levels of FAK were not affected by silencing of Itga5 and/or Itgb1 (Fig. 5B). Moreover, several specific cell signal pathway inhibitors, namely PD98059, U0126, LY294002, SB203580, SP600125, H-89, and AG490, were also added to the osteoblasts for 1 h, respectively, before ESWT. Both bands of total FAK and β-actin showed equal amounts of proteins subjected to protein electrophoresis (C). The data indicated that both PD98059 and U0126, unlike other inhibitors listed, inhibited ESW-induced FAK phosphorylation at Tyr-397 (D). Data represent the mean ± S.E. (error bars) in triplicate independent experiments (n = 3). The values of the control group were normalized to 100%. a, p > 0.05; b, p < 0.05; c, p < 0.01 as compared with the control group at the same time. d, p < 0.01; e, p > 0.05 versus ESW group.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Phospho-proteomics, Control, Western Blot, Expressing, Protein Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Enhancement of β-catenin activity by ESW (500 impulses at 10 kV) stimulation after elevation of integrin α5 and β1 expression. ESW raised β-catenin phosphorylation in 3 h (A). ERK1/2 inhibitor U0126 did not alter the activation of β-catenin (B). Cytosolic extracts of osteoblasts treated with ESW in the presence of U0126 (with a final concentration of 20 μm) for 60 min prior to ESW were subjected to Western blotting. Phosphorylated β-catenin and β-catenin were probed with anti-phospho-β-catenin and β-catenin primary monoclonal antibodies, respectively. C, note that in comparison with the control, ESW exposure markedly elevated the activation of β-catenin. Further studies on the relationship between expression of integrins and β-catenin by transfection have shown that knocking out integrins led to base-line level expression of activation of β-catenin (B and D). D, summary of the results (mean ± S.E. (error bars), n = 4, triplicate in each experiment). a, p > 0.05 as compared with the control at the same period. b, p < 0.01 as compared with the control at the same period. c, p > 0.05 as compared with the ESW group. d, p < 0.01 as compared with the ESW group.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Activity Assay, Expressing, Phospho-proteomics, Activation Assay, Concentration Assay, Western Blot, Bioprocessing, Comparison, Control, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Hypothetical model elucidating the regulation of phosphorylated FAK expression through an integrin α5 and β1-mediated MEK-ERK1/2-dependent pathway after ESWT. ESW directly stimulates integrin α5 and β1 mRNA expression inside the cell nucleus, and then the integrin protein expression is increased. Integrins induce MEK1/2 to phosphorylate ERK1/2, and then the activated ERK1/2 phosphorylates FAK and enhances its binding to the corresponding sites located in the adhesion sites, finally resulting in the enhancement of adhesion and migration. The Wnt/β-catenin signal pathway may also be involved in ESW-induced integrin-FAK signaling. The conformational activation of existing integrin α5/β1 complexes at the osteoblast surfaces may also be an additional potential mechanism that requires our further study.

Article Snippet: Cell Cultures Primary rat osteoblasts were isolated from calvaria of 3-day-old Sprague-Dawley rats and cultured using methods described previously ( 25 , 26 ).

Techniques: Expressing, Binding Assay, Migration, Activation Assay

Journal: Bone

Article Title: Ex Vivo Construction of Human Primary 3D-Networked Osteocytes

doi: 10.1016/j.bone.2017.09.012

Figure Lengend Snippet: [32] (a) microfluidic perfusion device with 6 culture chambers, (b) cross-sectional view of a 3D culture chamber with the red arrows indicating the overall direction of culture medium flow through the device, (c) schematic illustration of microbeads-guided assembly, and (d) histologic image of 3D-networked osteocytes with the red arrows indicating medium flow direction with respect to the tissue sample. Scale bar: 20 µm.

Article Snippet: A human 3D bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via: (1) the biomimetic assembly of primary human osteoblastic cells with 20–25 μm and BCP microbeads and (2) subsequent microfluidic perfusion culture.

Techniques:

Journal: Bone

Article Title: Ex Vivo Construction of Human Primary 3D-Networked Osteocytes

doi: 10.1016/j.bone.2017.09.012

Figure Lengend Snippet: (a) hip fragment shown as an example; (b) as-isolated cells after 4 collagenase digestion cycles; (c) proliferated osteoblastic cells after 10 days of 2D culture; (d) 3D tissue sample constructed using 20–25 µm microbeads and proliferated cells and 14 days of perfusion culture; (e) H&E histologic images showing the formation of 3D cellular network as indicated by black arrows in (f) and white arrows in (g); and (h) immunostaining for sclerostin (red). (d) –(f) from patient sample #6 and (g)–(h) from patient sample #4. Scale bar: 25 µm.

Article Snippet: A human 3D bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via: (1) the biomimetic assembly of primary human osteoblastic cells with 20–25 μm and BCP microbeads and (2) subsequent microfluidic perfusion culture.

Techniques: Isolation, Construct, Immunostaining