Journal: International Journal of Molecular Sciences

Article Title: Leptin Induces Oncostatin M Production in Osteoblasts by Downregulating miR-93 through the Akt Signaling Pathway

doi: 10.3390/ijms150915778

Figure Lengend Snippet: Leptin induces OSM expression in human osteoblasts. ( A , B ) Osteoblasts were incubated with various concentrationsof leptin for 24 h.Media and total RNA were collected, and the expression of OSM was examined by qPCR and ELISA assay ( n = 5); ( C , D ) Osteoblasts were incubated with leptin (30 nM) for 6, 12 or 24 h. Media and total RNA were collected, and the expression of OSM was examined by the qPCR and ELISA assay ( n = 4); ( E ) Osteoblasts from OA patients were incubated with various concentrationsof leptin for 24 h.Total RNA was collected, and the expression of OSM was examined by qPCR ( n = 3). The results are expressed as the mean ± SEM; * p < 0.05 as compared with the basal level.

Article Snippet: Human primary osteoblasts were obtained from Lonza (Walkersville, MD, USA), and the cells were maintained at 37 °C in 5% CO2 atmosphere in RPMI-1640 medium supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Leptin Induces Oncostatin M Production in Osteoblasts by Downregulating miR-93 through the Akt Signaling Pathway

doi: 10.3390/ijms150915778

Figure Lengend Snippet: Leptin induces OSM expression through the OBRl receptor. ( A ) Osteoblasts were transfected with OBRl and OBRs antisense oligonucleotide (AS-ODN) or OBRl and OBRs missense (MM)-ODN, and the mRNA level of OBRl and OBRs was analyzed by qPCR ( n = 5); ( B , C ) Osteoblasts were transfected with OBRl and OBRs AS-ODN or OBRl and OBRs MM-ODN for 24 h and then stimulated with leptin (30 nM) for 24 h; OSM expression was examined by the qPCR and ELISA assay ( n = 5). Results are expressed as the mean ± SEM; * p < 0.05 as compared with the basal level; # p < 0.05 as compared with the leptin-treated group.

Article Snippet: Human primary osteoblasts were obtained from Lonza (Walkersville, MD, USA), and the cells were maintained at 37 °C in 5% CO2 atmosphere in RPMI-1640 medium supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Leptin Induces Oncostatin M Production in Osteoblasts by Downregulating miR-93 through the Akt Signaling Pathway

doi: 10.3390/ijms150915778

Figure Lengend Snippet: Leptin increases OSM expression through inhibition of miR-93 expression.( A ) Osteoblasts were incubated with leptin (30 nM) for 24 h; the miRNAs’ expression was assessed by qPCR; ( B ) Osteoblasts were incubated with leptin for 24 h; miR-93’s expression was assessed by qPCR; ( C , D ) Osteoblasts were transfected with the miR-93 mimic for 24 h, followed by stimulation with leptin (30 nM) for 24 h; OSM expression was examined by the qPCR and ELISA assay ( n = 5). The results are expressed as the mean ± SEM; * p < 0.05 as compared with the basal level; # p < 0.05 as compared with the leptin-treated group.

Article Snippet: Human primary osteoblasts were obtained from Lonza (Walkersville, MD, USA), and the cells were maintained at 37 °C in 5% CO2 atmosphere in RPMI-1640 medium supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Inhibition, Incubation, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Leptin Induces Oncostatin M Production in Osteoblasts by Downregulating miR-93 through the Akt Signaling Pathway

doi: 10.3390/ijms150915778

Figure Lengend Snippet: Leptin increases OSM expression through the Akt pathway in osteoblasts. ( A – D ) Osteoblasts were pretreated with Akt inhibitor (10 µM) for 30 min or transfected with Akt siRNA for 24 h followed by stimulation with leptin (30 nM) for 24 h; OSM expression was examined by the qPCR and ELISA assay; ( E ) Osteoblasts were incubated with leptin (30 nM) for the indicated time intervals, Akt phosphorylation was examined by western blotting. Results are expressed as the mean ± SEM; * p < 0.05 as compared with the basal level; # p < 0.05 as compared with the leptin-treated group.

Article Snippet: Human primary osteoblasts were obtained from Lonza (Walkersville, MD, USA), and the cells were maintained at 37 °C in 5% CO2 atmosphere in RPMI-1640 medium supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Incubation, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Leptin Induces Oncostatin M Production in Osteoblasts by Downregulating miR-93 through the Akt Signaling Pathway

doi: 10.3390/ijms150915778

Figure Lengend Snippet: Leptin increases OSM expression by inhibition miR-93 through the Akt pathway.Osteoblasts were pretreated with Akt inhibitor (10 µM) ( A ) for 30 min or transfected with Akt siRNA ( B ) for 24 h followed by stimulation with leptin (30nM) for 24 h; miR-93 expression was measured by qPCR. Results are expressed as the mean ± SEM; * p < 0.05 as compared with the basal level; # p < 0.05 as compared with the leptin-treated group.

Article Snippet: Human primary osteoblasts were obtained from Lonza (Walkersville, MD, USA), and the cells were maintained at 37 °C in 5% CO2 atmosphere in RPMI-1640 medium supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Inhibition, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Leptin Induces Oncostatin M Production in Osteoblasts by Downregulating miR-93 through the Akt Signaling Pathway

doi: 10.3390/ijms150915778

Figure Lengend Snippet: Schema of signaling pathways involved in leptin-induced OSM expression in osteoblasts.Leptin enhances OSM production in human osteoblasts by inhibition miR-93 expression through the Akt signaling pathway.

Article Snippet: Human primary osteoblasts were obtained from Lonza (Walkersville, MD, USA), and the cells were maintained at 37 °C in 5% CO2 atmosphere in RPMI-1640 medium supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA).

Techniques: Protein-Protein interactions, Expressing, Inhibition

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: 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

Journal: Clinical Oral Investigations

Article Title: Backscatter from therapeutic doses of ionizing irradiation does not impair cell migration on titanium implants in vitro

doi: 10.1007/s00784-023-05128-6

Figure Lengend Snippet: Relative DNA damage with %DNA in tail normalized to the unirradiated samples on corresponding surface (C − ) and positive TCP control (C + ; 3% H 2 O 2 ), of primary human osteoblasts (OBs) and human mesenchymal stem cells (MSCs) immediately after irradiation with 0, 2, 6, and 10 Gy, while growing on tissue culture polystyrene (TCP), minimally rough machined titanium (Ti), or moderately rough fluoride-modified titanium (TiF) ( n = 3). Data are presented as mean ± SEM. * p ≤ 0.05 compared to corresponding 0 Gy control (C. − )

Article Snippet: Commercially available primary human osteoblasts (OBs; batch#: 0000426160) and human mesenchymal stem cells (MSCs; batch#: 0000684888) (Lonza, Walkersville, MD, USA) were routinely cultured at 37 °C/5% CO 2 in osteoblast basal medium (C-27010) supplemented with Osteoblast Growth Medium SupplementMix (C-39615) (Promocell, Heidelberg, Germany), or in human Mesenchymal Stem Cell Growth BulletKit Medium (MSCGM catalog no. PT-3001) (Lonza) respectively, both containing L-glutamine, gentamicin sulfate-amphotericin (GA), and 10% fetal bovine serum (FBS).

Techniques: Control, Irradiation, Modification

Journal: Clinical Oral Investigations

Article Title: Backscatter from therapeutic doses of ionizing irradiation does not impair cell migration on titanium implants in vitro

doi: 10.1007/s00784-023-05128-6

Figure Lengend Snippet: Representative images of primary human osteoblasts (OBs) migration and gap closure (GC) on tissue culture polystyrene (TCP), minimally rough machined titanium (Ti), and moderately rough fluoride-modified titanium (TiF), after no irradiation and 14 Gy (TCP) or 10 Gy (Ti + TiF), at the timepoints 0, 24, 48, and 72 h. As full gap closure was already observed at 48 h for all TCP samples, no imaging was performed at later timepoints. Scale bar: 200 µm

Article Snippet: Commercially available primary human osteoblasts (OBs; batch#: 0000426160) and human mesenchymal stem cells (MSCs; batch#: 0000684888) (Lonza, Walkersville, MD, USA) were routinely cultured at 37 °C/5% CO 2 in osteoblast basal medium (C-27010) supplemented with Osteoblast Growth Medium SupplementMix (C-39615) (Promocell, Heidelberg, Germany), or in human Mesenchymal Stem Cell Growth BulletKit Medium (MSCGM catalog no. PT-3001) (Lonza) respectively, both containing L-glutamine, gentamicin sulfate-amphotericin (GA), and 10% fetal bovine serum (FBS).

Techniques: Migration, Modification, Irradiation, Imaging