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

Journal: Biology

Article Title: FAM98 Family Proteins Play Distinct Roles in Osteoclastogenesis and Bone Resorption

doi: 10.3390/biology14010045

Figure Lengend Snippet: The key reagents used in this study.

Article Snippet: Antibodies , Mouse monoclonal anti-Nfatc1 , Santa Cruz Biotechnology, Dallas, TX, USA , sc-7294 , WB 1:250.

Techniques: Red Blood Cell Lysis, Plasmid Preparation, Transduction, Transfection, Staining, Lysis, Protease Inhibitor, Purification, Gene Expression, Quantitative RT-PCR

Journal: iScience

Article Title: Peroxiredoxin 1-Toll-like receptor 4-p65 axis inhibits receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation.

doi: 10.1016/j.isci.2024.111455

Figure Lengend Snippet: Figure 2. Gene profiling analysis of PRDX1 during osteoclastogenesis (A) The period for osteoclast differentiation was divided into four distinct time periods (T1 to T4). PRDX1 (200 nM) was treated during each period, and TRAP- positive multinucleated cells were counted. (B) Heatmap shows mRNA expression profiles of PRDX1 during osteoclastogenesis. K-means (K = 5) clustering of 4,328 differentially expressed genes (DEGs) in any pairwise comparison among four conditions (left panel). The clusters are indicated on the left. The right panel shows a heatmap showing the P-value sig- nificance and gene numbers of gene ontology (GO) analysis for genes in each cluster. (C) GSEA plot shows the increased gene expression of cell surface toll-like receptor signaling pathway in PRDX1 (PRDX1 + no RANKL condition) compared to control (no PRDX1 + no RANKL). Cfb and Ccl5 expression levels were measured by RT-qPCR using specific primers. (D) GSEA plot shows the decreased gene expression of the regulation of osteoclast differentiation in PRDX1 + RANKL treatment compared to RANKL treatment. mRNA expression levels of Ctsk, Dcstamp, and Nfatc1 were measured by RT-qPCR. The data are presented as the mean ± SD values from three independent experiments; P-value was examined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-NFATc1 Santa Cruz Cat# sc-7294; RRID:AB_2152503 Rabbit monoclonal anti-Cathepsin K Cell signaling Cat#57056; RRID:AB_3096128 Mouse monoclonal anti-DC-STAMP Millipore Cat#MABF39-I; RRID:AB_10807703 Mouse monoclonal anti-b2 Tubulin Santa Cruz Cat#SC-47751; RRID:AB_2210539 Rabbit monoclonal anti-NFkB p65 Cell signaling technoloy Cat# 8242; RRID:AB_3099658 Mouse monoclonal anti-b-Actin Sino Biological Cat# 100166-MM10; RRID:AB_2860060 Mouse monoclonal anti-LaminA/C Santa Cruz Cat# sc-376248; RRID:AB_10991536 Anti-TLR4 Protein Tech Cat# 19811-1-AP; PR1D:AB_10638446 Rabbit polyclonal anti- NFkB p65 Diagenode Cat# C15310256; RRID:AB_2721009 (ChIP) Chemicals, peptides, and recombinant proteins M-CSF This paper N/A RANKL This paper N/A Endotoxin Removal resin Thermofisher scientific Cat# 88270 EZ-Cytox DoGenBio Cat# EZ-3000 Tri-RNA reagent Favorgen Cat# FATRR001 M-MLV Reverse transcriptase Promega Cat# M1708 iQ SYBR Green Supermix Bio-rad Cat# 1708882 TAK242 Sigmaaldrich Cat# 614316 MyD inhibitor InvivoGen Cat# tlrl-pimyd Critical commercial assays Acid phosphatase Leukocyte (TRAP) kit Sigma-Aldrich Cat# 386A ATP Assay System Bioluminescence Detection Kit Promega Cat# FF2000 Deposited data RNA-seq data This paper GEO: GSE267039 Experimental models: Cell lines Mouse: WT ICR Raonbio Mouse: WT C57BL/6J Raonbio Mouse: Prdx1 / Gift from Dr. Dae-Yeul Yu (Ref) N/A Oligonucleotides Primers, see Table S3 This paper N/A Recombinant DNA pET15b-PRDX1 WT This paper N/A pET15b-PRDX1 C83S This paper N/A pET15b-PRDX1 4CS This paper N/A Software and algorithms Prism 10 GraphPad software RRID:SCR_002798 HOMER http://homer.ucsd.edu/homer/ Metascape http://metascape.org TRRUST https://www.grnpedia.org/trrust/ Biorender https://www.biorender.com/ Bioluminate https://www.schrodinger.com/ platform/products/bioluminate/ iScience 27, 111455, December 20, 2024 e1

Techniques: Expressing, Comparison, Gene Expression, Control, Quantitative RT-PCR

Journal: iScience

Article Title: Peroxiredoxin 1-Toll-like receptor 4-p65 axis inhibits receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation.

doi: 10.1016/j.isci.2024.111455

Figure Lengend Snippet: Figure 4. p65 is the transcription factor for PRDX1-TLR4 signaling (A) The most significantly enriched transcription factors regulating PRDX1 target genes belonging to cluster 4 (Figure 2B) using TRRUST v2. (B) Saa3 and Acod1 expression levels were measured from OCP cells treated with JSH23 (20 mM) in the presence of PRDX1 (200 nM). (C) OCP cells were treated with PRDX1 (200 nM) at the indicated times. Nuclear translocation of p65 was analyzed by immunoblotting with p65 antibody. The immunoblot band was quantified using ImageJ software and normalized to lamin C band intensity. (D) ChIP assay of p65 occupancy at the Saa3 and Acod1 promoters after 30 min of PRDX1 (200 nM) treatment. (E) OCP cells were treated with a combination of PRDX1 (200 nM) and RANKL (100 ng/mL) as indicated. Nuclear extracts were analyzed by immunoblotting for p65. The immunoblot band intensity was quantified using ImageJ software and normalized to lamin C band intensity. (F) ChIP assay of p65 occupancy at the Saa3, Acod1, and Nfatc1 promoters with a combination of PRDX1 (200 nM) and RANKL (100 ng/mL) for 30 min. (G) mRNA expression levels of Saa3, Acod1, and Nfatc1 treated with PRDX1 (100 and 200 nM) and/or RANKL (100 ng/mL) for 1 day. The data were presented as Mean ± SD from three independent experiments. p value is determined by a two-tailed t-test in (D) and one-way ANOVA with Tukey’s multiple comparisons test in (B, C, E, and, F). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-NFATc1 Santa Cruz Cat# sc-7294; RRID:AB_2152503 Rabbit monoclonal anti-Cathepsin K Cell signaling Cat#57056; RRID:AB_3096128 Mouse monoclonal anti-DC-STAMP Millipore Cat#MABF39-I; RRID:AB_10807703 Mouse monoclonal anti-b2 Tubulin Santa Cruz Cat#SC-47751; RRID:AB_2210539 Rabbit monoclonal anti-NFkB p65 Cell signaling technoloy Cat# 8242; RRID:AB_3099658 Mouse monoclonal anti-b-Actin Sino Biological Cat# 100166-MM10; RRID:AB_2860060 Mouse monoclonal anti-LaminA/C Santa Cruz Cat# sc-376248; RRID:AB_10991536 Anti-TLR4 Protein Tech Cat# 19811-1-AP; PR1D:AB_10638446 Rabbit polyclonal anti- NFkB p65 Diagenode Cat# C15310256; RRID:AB_2721009 (ChIP) Chemicals, peptides, and recombinant proteins M-CSF This paper N/A RANKL This paper N/A Endotoxin Removal resin Thermofisher scientific Cat# 88270 EZ-Cytox DoGenBio Cat# EZ-3000 Tri-RNA reagent Favorgen Cat# FATRR001 M-MLV Reverse transcriptase Promega Cat# M1708 iQ SYBR Green Supermix Bio-rad Cat# 1708882 TAK242 Sigmaaldrich Cat# 614316 MyD inhibitor InvivoGen Cat# tlrl-pimyd Critical commercial assays Acid phosphatase Leukocyte (TRAP) kit Sigma-Aldrich Cat# 386A ATP Assay System Bioluminescence Detection Kit Promega Cat# FF2000 Deposited data RNA-seq data This paper GEO: GSE267039 Experimental models: Cell lines Mouse: WT ICR Raonbio Mouse: WT C57BL/6J Raonbio Mouse: Prdx1 / Gift from Dr. Dae-Yeul Yu (Ref) N/A Oligonucleotides Primers, see Table S3 This paper N/A Recombinant DNA pET15b-PRDX1 WT This paper N/A pET15b-PRDX1 C83S This paper N/A pET15b-PRDX1 4CS This paper N/A Software and algorithms Prism 10 GraphPad software RRID:SCR_002798 HOMER http://homer.ucsd.edu/homer/ Metascape http://metascape.org TRRUST https://www.grnpedia.org/trrust/ Biorender https://www.biorender.com/ Bioluminate https://www.schrodinger.com/ platform/products/bioluminate/ iScience 27, 111455, December 20, 2024 e1

Techniques: Expressing, Translocation Assay, Western Blot, Software, Two Tailed Test

Journal: iScience

Article Title: Peroxiredoxin 1-Toll-like receptor 4-p65 axis inhibits receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation.

doi: 10.1016/j.isci.2024.111455

Figure Lengend Snippet: Figure 6. Model represents PRDX1-TLR4 signaling redirecting p65 to the Saa3 and Acod1 promoters rather than the Nfatc1 promoter (A) RANKL-RANK signaling pathway: RANKL binding to RANK activates p65, which translocates to the nucleus and binds to the Nfatc1 promoter, inducing osteoclastogenesis. (B) PRDX1-TLR4 signaling pathway: Activation of PRDX1-TLR4 signaling redirects p65 to the promoter regions of Saa3 and Acod1 instead of Nfatc1. As a result, PRDX1 induces the expression of Saa3 and Acod1 during RANKL-mediated osteoclast differentiation. The increase in SAA3 and itaconate, produced by ACOD1, inhibits osteoclast differentiation.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-NFATc1 Santa Cruz Cat# sc-7294; RRID:AB_2152503 Rabbit monoclonal anti-Cathepsin K Cell signaling Cat#57056; RRID:AB_3096128 Mouse monoclonal anti-DC-STAMP Millipore Cat#MABF39-I; RRID:AB_10807703 Mouse monoclonal anti-b2 Tubulin Santa Cruz Cat#SC-47751; RRID:AB_2210539 Rabbit monoclonal anti-NFkB p65 Cell signaling technoloy Cat# 8242; RRID:AB_3099658 Mouse monoclonal anti-b-Actin Sino Biological Cat# 100166-MM10; RRID:AB_2860060 Mouse monoclonal anti-LaminA/C Santa Cruz Cat# sc-376248; RRID:AB_10991536 Anti-TLR4 Protein Tech Cat# 19811-1-AP; PR1D:AB_10638446 Rabbit polyclonal anti- NFkB p65 Diagenode Cat# C15310256; RRID:AB_2721009 (ChIP) Chemicals, peptides, and recombinant proteins M-CSF This paper N/A RANKL This paper N/A Endotoxin Removal resin Thermofisher scientific Cat# 88270 EZ-Cytox DoGenBio Cat# EZ-3000 Tri-RNA reagent Favorgen Cat# FATRR001 M-MLV Reverse transcriptase Promega Cat# M1708 iQ SYBR Green Supermix Bio-rad Cat# 1708882 TAK242 Sigmaaldrich Cat# 614316 MyD inhibitor InvivoGen Cat# tlrl-pimyd Critical commercial assays Acid phosphatase Leukocyte (TRAP) kit Sigma-Aldrich Cat# 386A ATP Assay System Bioluminescence Detection Kit Promega Cat# FF2000 Deposited data RNA-seq data This paper GEO: GSE267039 Experimental models: Cell lines Mouse: WT ICR Raonbio Mouse: WT C57BL/6J Raonbio Mouse: Prdx1 / Gift from Dr. Dae-Yeul Yu (Ref) N/A Oligonucleotides Primers, see Table S3 This paper N/A Recombinant DNA pET15b-PRDX1 WT This paper N/A pET15b-PRDX1 C83S This paper N/A pET15b-PRDX1 4CS This paper N/A Software and algorithms Prism 10 GraphPad software RRID:SCR_002798 HOMER http://homer.ucsd.edu/homer/ Metascape http://metascape.org TRRUST https://www.grnpedia.org/trrust/ Biorender https://www.biorender.com/ Bioluminate https://www.schrodinger.com/ platform/products/bioluminate/ iScience 27, 111455, December 20, 2024 e1

Techniques: Binding Assay, Activation Assay, Expressing, Produced