Journal: International Journal of Molecular Medicine

Article Title: miR-181b-p53 negative feedback axis regulates osteosarcoma cell proliferation and invasion

doi: 10.3892/ijmm.2020.4558

Figure Lengend Snippet: miR-181b and p53 expression in OS tissues and cell lines The expression of (A) miR-181b and (B) p53 in noncancerous and OS tissue samples was determined by qPCR. (C) The correlation between miR-181b and p53 expression in OS tissue samples. The expression of (D) miR-181b and (E) p53 in normal HOBs and two OS cell lines U2OS and MG63 cells, was determined by qPCR. ** P<0.01. miR, microRNA; OS, osteosarcoma; NC, negative control; q, quantitative; HOBs, human osteoblasts.

Article Snippet: Primary human osteoblasts (HOBs) were obtained from Merck KGaA (cat. no. 406-05F) and cultured in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium (both from Gibco; Thermo Fisher Scientific, Inc.), with 2.5 mM L-glutamine (without phenol red) and 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Negative Control

Journal: PLoS ONE

Article Title: Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

doi: 10.1371/journal.pone.0048262

Figure Lengend Snippet: Dendrograms from unsupervised hierarchical clustering of the osteosarcoma cell lines, normal bone and normal osteoblast samples based on genome-wide (A) mRNA expression (vst transformed and quantile normalised probe intensities), (B) DNA methylation (probe beta values) and (C) DNA copy number (probe intensities). The osteosarcoma cell lines have been colour-coded in gray and black, highlighting the two main subclusters, the normal bone samples in red and the normal osteoblast samples in blue. The clusters were made using Spearman correlation as distance measure and complete linkage.

Article Snippet: Two primary osteoblast cultures isolated from human calvaria of different donors were purchased from ScienCell Research Laboratories (California, USA).

Techniques: Genome Wide, Expressing, Transformation Assay, DNA Methylation Assay

Journal: PLoS ONE

Article Title: Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

doi: 10.1371/journal.pone.0048262

Figure Lengend Snippet: Plot of (A) DNA copy number levels of DLX5 and RUNX2 based on quantitative real-time PCR and (B) mRNA expression levels of DLX5 and RUNX2 based on quantitative real-time RT-PCR, in five cell lines and five tumour samples. The DNA copy number levels have been normalised to the average copy number of two control genes, EEF1G and FBXO11 , whereas the mRNA expression levels have been normalised to the expression of the house-keeping gene GAPDH and then to the average expression level of the two normal osteoblast samples. The DNA copy number and mRNA expression levels based on the microarray data are indicated for the cell lines; N, normal copy number/expression; G, gain; O, over-expression.

Article Snippet: Two primary osteoblast cultures isolated from human calvaria of different donors were purchased from ScienCell Research Laboratories (California, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Control, Microarray, Over Expression

Journal: PLoS ONE

Article Title: Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

doi: 10.1371/journal.pone.0048262

Figure Lengend Snippet: Plot of the DNA methylation status and mRNA expression level of CXCL5 based on methylation-specific PCR and quantitative real-time RT-PCR, respectively, in five cell lines and five tumour samples. The mRNA expression levels have been normalised to the expression of the house-keeping gene GAPDH and then to the average expression level of the two normal osteoblast samples. The DNA methylation status is indicated with coloured circles; black, full methylation, grey, partial methylation. The DNA methylation and mRNA expression levels based on the microarray data are indicated for the cell lines; N, normal methylation; H, hyper-methylation; U, under-expression.

Article Snippet: Two primary osteoblast cultures isolated from human calvaria of different donors were purchased from ScienCell Research Laboratories (California, USA).

Techniques: DNA Methylation Assay, Expressing, Methylation, Quantitative RT-PCR, Microarray

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: 8-Nitro-cGMP suppresses mineralization by mouse osteoblasts

doi: 10.3164/jcbn.21-129

Figure Lengend Snippet: Effects of IL-1β and TNF-α on the production of 8-nitro-cGMP and expressions of osteoblast marker genes in mouse osteoblasts. (A) Mouse calvarial osteoblasts were exposed to IL-1β (1 ng/ml) and TNF-α (10 ng/ml) for 24 h in the absence (−) and presence (+) of l -NAME (5 mmol/L), after which 8-nitro-cGMP was detected immunocytochemically using a specific antibody (red). Bars, 100 μm. (B) The effects of the introduction of Nos2 siRNA and treatments with IL-1β (1 ng/ml) and TNF-α (10 ng/ml) on the expression of Nos2 mRNA in osteoblasts were examined by RT-PCR, with Gapdh expression used as a control. (C, D) The effects of the introduction of Nos2 siRNA and treatments with IL-1β (1 ng/ml) and TNF-α (10 ng/ml) on expressions of Tnap mRNA (C) and Bglap mRNA (D) in osteoblasts were evaluated by real-time RT-PCR, with the expression levels of those genes normalized to that of Gapdh . Results are expressed as relative to the control culture (far left columns). Values are shown as the mean ± SD of 4 cultures. *Significant difference ( p <0.05).

Article Snippet: Mouse primary osteoblasts were isolated from calvaria of newborn ddY mice provided by Japan SLC Inc. (Hamamatsu, Japan) using a conventional method, ( ) according to a protocol approved by the ethical board for animal experiments of Showa University (approval number 17055).

Techniques: Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: 8-Nitro-cGMP suppresses mineralization by mouse osteoblasts

doi: 10.3164/jcbn.21-129

Figure Lengend Snippet: Effects of exogenous 8-nitro-cGMP on the intracellular 8-nitro-cGMP and the formation of S -guanylated proteins in osteoblasts and the proliferation of osteoblasts. (A, B) Mouse primary osteoblasts were cultured for 48 h in the absence and presence of 30 μmol/L 8-nitro-cGMP. The intracellular 8-nitrocGMP was determined by LC-MS/MS. (A) Representative elution profiles of 8-nitro-cGMP of the extracts of the cells treated without (a) and with (b) exogenous 8-nitro-cGMP. (c) A representative chromatogram of the authentic [‍ 13 C 10 ]8-nitro-cGMP. (B) The amount of intracellular 8-nitro-cGMP in osteoblasts cultured for 48 h in the presence or absence of 30 μmol/L 8-nitro-cGMP. Values are shown as the mean ± SD ( n = 3). (C, D) Production of S -guanylated proteins in osteoblasts after treatment with 8-nitro-cGMP. Osteoblasts were treated for 12 h with the indicated concentrations of 8-nitro-cGMP. (C) Western blot analysis of S -guanylated proteins and b-actin in cell lysates was performed using the anti-RS-cGMP and anti-b-actin antibodies. (D) The intensity of S -guanylated proteins was normalized by β-actin. (E) Mouse primary osteoblasts were cultured for the indicated periods in the absence (dotted line) or presence of 8-nitro-cGMP (30 μmol/L) (solid line). The number of trypan blue-unstained cells was counted. Values are shown as the mean ± SD ( n = 4).

Article Snippet: Mouse primary osteoblasts were isolated from calvaria of newborn ddY mice provided by Japan SLC Inc. (Hamamatsu, Japan) using a conventional method, ( ) according to a protocol approved by the ethical board for animal experiments of Showa University (approval number 17055).

Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: 8-Nitro-cGMP suppresses mineralization by mouse osteoblasts

doi: 10.3164/jcbn.21-129

Figure Lengend Snippet: Effects of 8-nitro-cGMP and 8-bromo-cGMP on osteoblastic phenotypes in mouse osteoblasts. (A–C) ALP activity, mineralization, and expression of Bglap mRNA in osteoblasts cultured in the presence and absence of 8-nitro-cGMP (30 μmol/L) were determined. (A) ALP activity staining of mouse primary osteoblasts is shown in the upper photographs, while the lower panel shows specific ALP activities. (B) Mineralized nodules formed in cultures of MC3T3-E1 cells were stained with Alizarin red (upper photographs). Alizarin red bound to the extracellular matrix was determined by measuring the optical density after solubilization (lower panel). (C) The expression of Bglap mRNA in mouse primary osteoblasts was evaluated by real-time RT-PCR and normalized to Gapdh . Results are expressed as relative to the control culture. Values are shown as the mean ± SD of 4 cultures. (D, E) ALP activity in mouse primary osteoblasts (D) and mineralization by MC3T3-E1 cells (E) cultured in the presence or absence of 8-bromo-cGMP (30 μmol/L). Values are shown as the mean ± SD of 4 cultures. *Significant difference ( p <0.05). (A, B) Bars, 500 μm.

Article Snippet: Mouse primary osteoblasts were isolated from calvaria of newborn ddY mice provided by Japan SLC Inc. (Hamamatsu, Japan) using a conventional method, ( ) according to a protocol approved by the ethical board for animal experiments of Showa University (approval number 17055).

Techniques: Activity Assay, Expressing, Cell Culture, Staining, Quantitative RT-PCR, Control

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: 8-Nitro-cGMP suppresses mineralization by mouse osteoblasts

doi: 10.3164/jcbn.21-129

Figure Lengend Snippet: Effects of Cars2 siRNA on osteoblastic phenotypes in mouse osteoblasts. (A) The introduction of Cars2 significantly reduced the expression of Cars2 mRNA in mouse primary osteoblasts. (B) The amounts of S -guanylated proteins and b-actin in mouse primary osteoblasts were determined by Western blotting. The addition of Cars2 siRNA significantly increased the amounts of S -guanylated proteins in osteoblasts. (C–E) ALP activity, mineralization, and the expression of Bglap mRNA in mouse primary osteoblasts introduced with control siRNA or Cars2 siRNA were examined. (C) ALP activity staining is shown in the upper photographs, while the lower panel shows specific ALP activities. (D) Mineralized nodules were stained with Alizarin red (upper photographs). Alizarin red bound to the extracellular matrix was determined by measuring optical density after solubilization (lower panel). (C, D) Bars, 500 μm. (E) The expression of Bglap mRNA was evaluated by real-time RT-PCR. The level of Bglap expression was normalized to that of Gapdh and shown as relative to the control culture. Values are expressed as the mean ± SD of 4 cultures. *Significant difference ( p <0.05).

Article Snippet: Mouse primary osteoblasts were isolated from calvaria of newborn ddY mice provided by Japan SLC Inc. (Hamamatsu, Japan) using a conventional method, ( ) according to a protocol approved by the ethical board for animal experiments of Showa University (approval number 17055).

Techniques: Expressing, Western Blot, Activity Assay, Control, Staining, Quantitative RT-PCR