Journal: Journal of Bone Metabolism

Article Title: Effects of the Lysine Methyltransferase Inhibitor AZ505 on Bone Metabolism

doi: 10.11005/jbm.2021.28.4.297

Figure Lengend Snippet: AZ505 inhibits osteoclast differentiation in vitro . (A) Bone marrow-derived macrophages (BMMs) were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) with or without 1.2 μM AZ505 for 6 days and stained for tartrate-resistant acid phosphatase (TRAP) (×100 magnification). (B, C) BMMs were cultured in the presence of M-CSF and RANKL with or without AZ505 for the indicated days and subjected to reverse transcription-quantitative polymerase chain reaction. (D) Protein lysates were prepared from cells cultured for the indicated days with M-CSF and RANKL in the presence or absence of AZ505. Western blot was performed using antibodies for nuclear factor of activated T cells (NFATc1), c-Fos and β-actin. Data are presented as mean±standard deviation. NS, not significant. * P <0.05. ** P <0.005 vs. vehicle.

Article Snippet: Recombinant human macrophage colony-stimulating factor (M-CSF) and human soluble receptor activator of nuclear factor-κB ligand (RANKL) were purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: In Vitro, Derivative Assay, Cell Culture, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Journal of Bone Metabolism

Article Title: Effects of the Lysine Methyltransferase Inhibitor AZ505 on Bone Metabolism

doi: 10.11005/jbm.2021.28.4.297

Figure Lengend Snippet: Receptor activator of nuclear factor-κB ligand (RANKL) expression is induced by AZ505. (A–C) Calvarial preosteoblasts were cultured in osteogenic medium containing either vehicle or AZ505 (1.2 μM) for the indicated days. The mRNA levels of tnfsf11 and tnfsf11b were analyzed by reverse transcription-quantitative polymerase chain reaction and the RANKL to osteoprotegerin ratio was calculated. (D) Calvarial preosteoblasts and bone marrow cells were co-cultured for 10 days with 1.2 μM AZ505 or vehicle and stained for tartrate-resistant acid phosphatase (TRAP) (×100 magnification). TRAP + multinucleated osteoclasts were counted. Data are presented as mean±standard deviation. ** P <0.005 vs. vehicle.

Article Snippet: Recombinant human macrophage colony-stimulating factor (M-CSF) and human soluble receptor activator of nuclear factor-κB ligand (RANKL) were purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Expressing, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Standard Deviation

Journal: Research Square

Article Title: Long noncoding RNA Malat1 inhibits Tead3-Nfatc1–mediated osteoclastogenesis to suppress osteoporosis and bone metastasis

doi: 10.21203/rs.3.rs-2405644/v1

Figure Lengend Snippet: a-d. CD14 + human placental macrophages were differentiated into multinucleated giant cells (MGCs) in culture. Both CD14 + macrophages and MGCs were subjected to high-throughput RNA sequencing (RNA-seq). n = 6 biological replicates per group. Data source: GSE38747. a. Heatmap of differentially expressed genes between CD14 + macrophages and MGCs. b. Volcano plot of genes upregulated (red) or downregulated (blue) in MGCs relative to CD14 + macrophages. Cut-off values: |log 2 (fold change)| > 1 and P value < 0.001. c. Relative expression levels of MALAT1 and osteoclast markers were quantitated from the RNA-seq results. d. Gene set enrichment analysis (GSEA) of the RNA-seq data, showing the top 14 Gene Ontology (GO) pathways of 486 significant pathways. e-h. qPCR of Nfatc1 ( e ), Ctsk ( f ), Trap5 ( g ), and Malat1 ( h ) in RAW264.7 cells treated with soluble RANKL (50 ng/mL) for the indicated times. i. Schematic representation of the treatments used to evaluate the osteoclastogenic activity of RANKL, LPS, and TNF-α, with or without pretreatment (priming) with RANKL. j. TRAP staining images (left panel) and quantification (right panel) of RAW264.7 cells treated with RANKL or LPS, with or without pretreatment with RANKL. k. qPCR of Nfatc1 , Ctsk , and Malat1in the cells described in j . l. TRAP staining images (left panel) and quantification (right panel) of RAW264.7 cells treated with RANKL or TNF-α, with or without pretreatment with RANKL. m. qPCR of Nfatc1 , Ctsk , and Malat1in the cells described in l . Statistical significance in c and j-m was determined by a two-tailed unpaired t -test. Error bars are s.e.m.

Article Snippet: Cells were seeded in 24-well or 6-well plates at a density of 2 × 10 4 or 5 × 10 5 cells per well; 50 ng/mL of mouse soluble RANKL (Peprotech, 315-11) was used to induce differentiation, and the culture medium was changed every 2 days.

Techniques: High Throughput Screening Assay, RNA Sequencing, Expressing, Activity Assay, Staining, Two Tailed Test