Journal: Cell Death & Disease

Article Title: TSP-1 interaction with RANK and OPG: implications for bone remodeling and osteolytic bone metastasis

doi: 10.1038/s41419-026-08600-9

Figure Lengend Snippet: RAW 264.7 cells, pretreated with E123CaG (1 μM) in serum-free medium for 3 h were stimulated with RANKL (50 ng/ml) for 20 min ( A ) or 48 h ( B ). A Western blot analysis of phosphorylated p38 (p-38), total p38, phosphorylated JNK (p-JNK), and total JNK. Tubulin was used as a loading control. Graph, quantitative analysis. MAPK activation (phosphorylated protein/total protein ratio) is expressed as the percentage of controls, mean ± SEM of two independent experiments (* P < 0.05 t test). B Nuclear translocation of NFATc1. Representative images of cells treated (RANKL) or not (Control) with RANKL with or without E123CaG (blue, nuclei; red, NFATc1, scale bar 25 µm). Graph, NFATc1 distribution is expressed as a nuclear to cytoplasmic ratio. Data are mean ± SEM from one experiment representative of two (** P < 0.002; * P < 0.05 one-way ANOVA and Tukey’s).

Article Snippet: Cells were then stained with the anti-NFATc1 primary antibody (Sc-7294, Santa Cruz Biotechnology, Heidelberg, Germany 1:200) in PBS 1% BSA overnight at 4 °C, followed by donkey anti-mouse Cy3 secondary antibody for 1 h at room temperature.

Techniques: Western Blot, Control, Activation Assay, Translocation Assay

Journal: Science Advances

Article Title: Exogenous activation of the adhesion GPCR ADGRD1/GPR133 protects against bone loss by negatively regulating osteoclastogenesis

doi: 10.1126/sciadv.ads3829

Figure Lengend Snippet: ( A ) Intracellular cAMP levels in WT BMM cells after induction with RANKL (100 ng/ml), DMSO, or GL64 (30 μM). The data are presented as the means ± SD. ** P < 0.01. Two independent biological replicates were performed. ( B ) Intracellular cAMP levels in vector- and ADGRD1 -overexpressing RAW264.7 cells. The data are presented as the means ± SD. * P < 0.05. Four independent biological replicates were performed. ( C ) Representative images of TRAP staining of BMMs treated with DMSO, GL64 (10 μM), or H89 (1 μM) and stimulated with RANKL (100 ng/ml) for 6 days. Scale bars, 50 μm. ( D ) Quantification of osteoclast area and number shown in (C) (area: F 3,6 = 45.34, P = 0.0002; number: F 3,6 = 69.20, P < 0.0001). The data are presented as the means ± SD. ** P < 0.01 and *** P < 0.001. Three independent biological replicates were performed. ( E ) Representative TRAP staining images of WT and Adgrd1 −/− BMMs treated with RANKL (100 ng/ml) or IBMX (50 μM) for 6 days. Scale bars, 50 μm. ( F ) Quantification of the osteoclast area (top) and number (bottom) of the cells shown in (E) (area: F 3,6 = 30.08, P = 0.0005; number: F 3,6 = 291.4, P < 0.0001). The data are presented as the means ± SD. * P < 0.05 and *** P < 0.001. Three independent biological replicates were performed. ( G ) Representative images of NFATC1 nuclear translocation in BMM cells treated with DMSO, GL64 (10 μM), or H89 (1 μM) and stimulated with RANKL (100 ng/ml) for 2 days. Scale bars, 50 μm. ( H ) Percentage of NFATC1 nuclear localization in the samples shown in (G) ( F 3,6 = 34.52, P = 0.0004). Data are presented as the means ± SD. * P < 0.05. Three independent biological replicates were performed.

Article Snippet: After being removed from the medium and washed three times with PBS, the slides were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% bovine serum albumin, and then incubated with a mouse anti-NFATC1 primary antibody (Santa Cruz, Sc-7294, 1:200, US) for 24 hours at 4°C.

Techniques: Plasmid Preparation, Staining, Translocation Assay