Journal: Lipids

Article Title: Conjugated linoleic acid prevents ovariectomy-induced bone loss in mice by modulating both osteoclastogenesis and osteoblastogenesis

doi: 10.1007/s11745-013-3872-5

Figure Lengend Snippet: Eight weeks old C57BL/6 mice were ovariectomized or sham operated and fed experimental diets for 24 weeks. Thirty two weeks old mice were then sacrificed and splenocytes were isolated from spleen. (A) RNA was prepared, reverse transcribed and amplified by PCR using primers designed for genes of RANKL, OPG and GAPDH and visualized on agarose gels with ethidium bromide. (B) Relative expression of RANKL and OPG is shown. The intensity of the bands was determined by densitometry and normalized by the level of GAPDH. n=3 mice per group. P < 0.05 was considered significant. Flow cytometric analysis of RANKL expression in splenocytes (C, D and E). RANKL positive total splenocytes (C), RANKL positive CD8 T cells (D) and RANKL positive CD4 T cells (E) cells in splenocytes were analyzed for the expression of the cell surface determinants for RANKL only, CD8/RANKL and CD4/RANKL respectively. Before staining, FcγII/III receptors on cells were blocked by pre-incubation of the samples with a rat anti-CD32/16 Ab. Cells were then incubated with the anti-RANKL with or without anti-CD8 or anti-CD4 antibodies for 30 min at 4 °C. The cells were re-suspended in wash buffer and analyzed on a FACScan flow cytometer. n=3 mice per group. P value <0.05 was considered significant by student’s t test.

Article Snippet: Serum bone turnover markers measurement Bone resorption markers, receptor activator of NF-κB ligand (RANKL), and tartrate resistant acid phosphatase (TRAP)-5b and bone formation marker, N-terminal propeptide of type I procollagen (PINP) were measured in serum samples using mouse free soluble RANKL, mouse OPG, mouse TRAP5b, mouse PINP assay kits from Immunodiagnostic System (IDS) Inc. (Fountain Hills, AZ, USA) and bone formation marker, alkaline phosphatase (ALP) was measured in serum samples using Quantichrome alkaline phosphatase assay kit from Bioassay Systems (Hayward, CA, USA) according to the manufacturer’s instructions ( 17 ).

Techniques: Isolation, Reverse Transcription, Amplification, Expressing, Staining, Incubation, Flow Cytometry

Journal: Archives of Toxicology

Article Title: Establishment of a human 3D in vitro liver-bone model as a potential system for drug toxicity screening

doi: 10.1007/s00204-024-03899-9

Figure Lengend Snippet: The antibodies used for dot blot

Article Snippet: Soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) , 500-m46 , PeproTech, Hamburg, Germany , 1:1000.

Techniques:

Journal: Archives of Toxicology

Article Title: Establishment of a human 3D in vitro liver-bone model as a potential system for drug toxicity screening

doi: 10.1007/s00204-024-03899-9

Figure Lengend Snippet: DCFH-DA assay was used to detect the ROS levels in liver spheroids. Ellman assay was used to detect the GSH and GSSG levels in liver spheroids. The expression of secreted IL-6, sRANKL, and OPG protein levels in the liver-bone system supernatant were measured by dot blot. a After acute exposure to diclofenac, the ROS levels in liver spheroids on day 7, H 2 O 2 stimulation as a positive control. b After acute exposure to diclofenac, the ratio of GSH and GSSG in liver spheroids on day 7. c With the stimulation of 3–6 µM diclofenac, the IL-6 protein levels in supernatant on day 7, and the representative image of dot blot. d With the stimulation of 3–6 µM diclofenac, the sRANKL protein levels in supernatant on day 7, and the representative image of dot blot. e With the stimulation of 3–6 µM diclofenac, the OPG protein levels in supernatant on day 7, and the representative image of dot blot. f The ratio of sRANKL and OPG in the liver-bone co-culture system. The Kruskal–Wallis test followed by Dunn’s multiple comparison test was used to determine statistical differences. Data are presented as means ± SEM, and the significance is shown as * p < 0.05, *** p < 0.001, and **** p < 0.0001 vs. Control group. N ≥ 3, n ≥ 2

Article Snippet: Soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) , 500-m46 , PeproTech, Hamburg, Germany , 1:1000.

Techniques: DCFH-DA Assay, Expressing, Dot Blot, Positive Control, Co-Culture Assay, Comparison, Control

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