Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: Relative percentage of bases in each chromHMM ( ; ) category throughout the entire genome ( a ), in fixed or nearly fixed modern human-derived variants ( b ), in active sequences ( c ), and in differentially active sequences ( d ), per cell type. See Discussion for cell-type specificity and enhancer enrichment. ( e ) Histogram of the number of tissues and number of sequences with transcription start site- (TSS) or enhancer-related chromHMM marks for all 14,042 sequences. Tissues and cell types investigated include embryonic stem cells (ESCs), osteoblasts, neural progenitor cells (NPCs), mesenchymal stem cells, monocytes, skin fibroblasts, brain hippocampus, skeletal muscle, heart left ventricle, sigmoid colon, ovary, fetal lung, and liver. Inset shows data for ESC, osteoblast, and NPC only.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: Derivative Assay

Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: ( a ) Overlap between cell types of active sequences. Super Exact test p-value is shown for the overlap of the three groups. ( b-d ) Enrichment levels of active and repressive histone modification marks within active sequences. Enrichment is computed compared to inactive sequences. The enrichment of H3K27me3 in embryonic stem cells (ESCs) possibly reflects the presence of this mark in bivalent genes, which become active in later stages of development . For confidence intervals, see . ( e ) Enrichment of differentially active sequences in various chromatin-based genomic annotations. Missing circles reflect no differentially active sequences in that category. Stars mark significant enrichments (false discovery rate [FDR] <0.05). ( f ) Violin plots of DNA methylation levels for active (green) vs. inactive (red) sequences in osteoblasts. Methylation levels per sequence were computed as the mean methylation across all modern and archaic human bone methylation samples. The circle marks mean methylation across all sequences in each group. t -test p-value is shown.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: Modification, DNA Methylation Assay, Methylation, Sequencing

Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: ( a–c ) Violin plots of DNA methylation levels in modern and archaic human bone methylation samples, for differentially active ( a ), promoter differentially active ( b ), and CpG-poor promoter differentially active ( c ) sequences in osteoblasts. Promoter sequences are sequences between 5 kb upstream and 1 kb downstream of a transcription start site (TSS). CpG-poor promoter sequences were defined as the bottom 50% promoter sequences. ( d ) Violin plots of absolute predicted TF binding score difference between modern and archaic sequences. Points show mean.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: DNA Methylation Assay, Methylation, Binding Assay

Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: (a–c) Expression fold-change vs. predicted TF binding fold-change for each sequence. Positive scores represent increased binding in the modern sequence. Parentheses show number of points in each quadrant with a score difference >0. ( d ) Pearson’s correlation between differential expression and predicted differential binding affinity. Only significant TFs (false discovery rate [FDR] ≤0.05, ) are shown for osteoblasts (yellow) and neural progenitor cells (NPCs) (red). ( e ) Expression fold-change vs. predicted TF binding fold-change for ZNF281 in NPCs. Pearson’s r and p-value are shown. ( f ) Enriched Gene Ontology terms for embryonic stem cells (ESCs) (blue), osteoblasts (yellow), and NPCs (red). ( g ) Expression fold-change of differentially active sequences compared to the cis -regulatory expression fold-change between human and chimpanzee of genes associated with these sequences. cis -regulatory expression changes were taken from hybrid human-chimpanzee induced pluripotent stem cells (iPSCs) . ( h ) RT-qPCR validation of NPCs at passage 1 (pink) and passage 10 (red). Expression levels are normalized to HPRT expression.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: Expressing, Binding Assay, Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Biomarker Discovery

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: 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