Journal: International Journal of Biological Sciences

Article Title: GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

doi: 10.7150/ijbs.70620

Figure Lengend Snippet: Gpr125 is expressed in mice osteoclasts in vitro and in vivo and increases during the differentiation from monocytes to osteoclasts. (A) Heatmap analysis of 20 GPCR mRNA expression levels in monocytes and osteoclasts by RNA-seq analysis. Orange denotes high expression; blue denotes low expression. (B) Column chart analysis of the fold change of 20 GPCR mRNA expression levels when cells differentiated from monocytes to osteoclasts from RNA-seq analysis. (C) Protein level of GPR125 in macrophages and osteoclasts induced for 5 days. (D) Quantification of C. (E) Gpr125 expression in osteoclast of the femur sample of newborn mouse in vivo showed by IF stain with anti-Gpr125 (red), anti-Ctsk (green) and DAPI (blue). Scale bar, 400μm. Bars on graphs represent mean ± SEM, * p < 0.05. ns, not significant.

Article Snippet: The GPR125 polyclonal antibody (PA5-99891) was from Invitrogen.

Techniques: In Vitro, In Vivo, Expressing, RNA Sequencing Assay, Staining

Journal: International Journal of Biological Sciences

Article Title: GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

doi: 10.7150/ijbs.70620

Figure Lengend Snippet: Knockdown of Gpr125 impairs osteoclast differentiation and function. (A-G) MBM was transduced by sh-SC, sh-GPR125 lentivirus, or mock control and stimulated by M-CSF (10ng/mL) and RANKL (10ng/mL). (A) When cells were induced by M-CSF/RANKL for 5 days, cells were stained for TRAP activity at a 12-well-plate. Scale bar = 200μm. (B) To detect the acid secretion function, cells were performed with Acridine Orange (AO) stain in a 24-well-plate when induced by M-CSF/ RANKL for 5 days. Scale bar = 200μm. (C) To determine the bone resorption activity of mature osteoclasts, cells of three groups were stained with WGA reagent. Scale bar = 100μm. (D) Quantification of A-C . (E) Western blot analysis of osteoclasts from three groups on Day 5 to confirm the knockdown of Gpr125 . (F) Quantification of protein levels of Gpr125 in E normalized to Gapdh. (G) Western blot analysis of osteoclasts from three groups on Day 5 to explore the influence of the knockdown of Gpr125 on the expression of osteoclast marker protein Pu.1. (H) Quantification of protein levels of Gpr125 and Pu.1 in G normalized to Gapdh. Data is expressed as mean ± SEM, one dot represents one sample (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The GPR125 polyclonal antibody (PA5-99891) was from Invitrogen.

Techniques: Staining, Activity Assay, Western Blot, Expressing, Marker

Journal: International Journal of Biological Sciences

Article Title: GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

doi: 10.7150/ijbs.70620

Figure Lengend Snippet: Knockdown of Gpr125 inhibits F-Actin ring formation and decreases osteoclastogenesis-related genes expressions. (A-C) MBMs infected by different lentivirus respectively and induced by M-CSF (10 ng/mL) and RANKL (10 ng/mL). (A) To detect the influence of Gpr125 knockdown on F-Actin ring formation, cells cultured for 5 days were conducted with F-Actin ring stain. DAPI (blue), F-Actin (red), merge of F-Actin and DAPI (purple). Scale bar = 100μm. (B) Quantification of F-actin ring positive cells in A. (C) qRT-PCR of the sh-GPR125 treated osteoclasts to detect mRNA expression levels of Gpr125 , Ctsk , Nfatc1 , Acp5 , and Atp6i compared with GAPDH . One dot represents one sample (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, no significance.

Article Snippet: The GPR125 polyclonal antibody (PA5-99891) was from Invitrogen.

Techniques: Infection, Cell Culture, Staining, Quantitative RT-PCR, Expressing

Journal: International Journal of Biological Sciences

Article Title: GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

doi: 10.7150/ijbs.70620

Figure Lengend Snippet: Overexpression of Gpr125 increases osteoclastogenesis related gene expression at the mRNA level. (A) Western blot analysis confirming the overexpression of Gpr125 after cells were transfected with pLX304-GPR125. (B) The quantification of data of (A). (C) qRT-PCR results of the pLX304-GPR125 infected osteoclasts. When cells were cultured for 5 days, qRT-PCR was carried out to measure the mRNA expression levels of Gpr125 , Ctsk , Acp5 , and Atp6i normalized by GAPDH . * p < 0.05, ** p < 0.01.

Article Snippet: The GPR125 polyclonal antibody (PA5-99891) was from Invitrogen.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Infection, Cell Culture

Journal: International Journal of Biological Sciences

Article Title: GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

doi: 10.7150/ijbs.70620

Figure Lengend Snippet: Overexpression of Gpr125 promotes osteoclast differentiation and function. (A-D) Overexpression of Gpr125 increases osteoclast differentiation. (A-G) MBMs were transduced by pLX304 or pLX304-GPR125 lentiviruses, and cells were stimulated by M-CSF (10 ng/mL) and RANKL (10 ng/mL). (A) When cells were cultured for 5 days, TRAP stain was performed to show TRAP activity. Scale bar = 200 μm. (B) After inducing cells by both M-CSF and RANKL for 5 days, AO stain was conducted to detect their acid secretion performance. Scale bar = 200 μm. (C) Cells seeded in bone slides were cultured for 5 days and underwent TRAP stain to show their TRAP activity when grown physiologically. Scale bar = 100 μm. (D) WGA stain was conducted to determine bone resorption activity when cells were cultured in bone slides for 7 days. Scale bar = 100 μm. (E-F) To detect the F-Actin ring formation and Ctsk expression levels at the same time, cells were seeded into wells (E) and bone slides (F) then cultured for 5 days. F-Actin stain and anti-Ctsk IF stain were performed simultaneously. DAPI (blue), F-Actin ring (red), anti-Ctsk (green), and merge region (orange). The frame region in merge picture of each group was shown in the boxed area. (G) Quantification of (A-F) . TRAP positive MNCs (Multinucleated cells) (≥ 3 nuclei). One dot represents one sample (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The GPR125 polyclonal antibody (PA5-99891) was from Invitrogen.

Techniques: Over Expression, Cell Culture, Staining, Activity Assay, Expressing

Journal: International Journal of Biological Sciences

Article Title: GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

doi: 10.7150/ijbs.70620

Figure Lengend Snippet: Gpr125 promotes osteoclastogenesis via NF-κB and MAPK signaling pathways. (A) MBMs infected by pLX304 or pLX304-GPR125 viruses, respectively, were continuously stimulated by M-CSF (10ng/mL)/RANKL (10ng/mL) for 5 days. Western blot analysis was used to confirm the overexpression efficiency of Gpr125 and to detect the expression levels of NFATc1, CTSK, using Tubulin as control. (B) Quantification data of (A) . Protein expression levels were normalized by GAPDH . (C) Western blot analysis was used to explore the change of the phosphorylation of ERK, JNK, and p38 between sh-GPR125 and sh-NC groups, using GAPDH as the control. These cells were stimulated by RANKL (20ng/mL) when induced by M-CSF (10ng/mL) and RANKL (10ng/mL) for 3 days. (D) Quantification data of (C) . (E) Western blot analysis was used to explore the change of the phosphorylation of p65, IKBα, and AKT between sh-GPR125 and sh-NC groups. (F) Quantification data of (E) . The phosphorylation levels were shown by phosphor-protein level versus total protein level. One dot represents one sample (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, no significance.

Article Snippet: The GPR125 polyclonal antibody (PA5-99891) was from Invitrogen.

Techniques: Infection, Western Blot, Over Expression, Expressing

Journal: International Journal of Biological Sciences

Article Title: GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

doi: 10.7150/ijbs.70620

Figure Lengend Snippet: The working model of how GPR125 positively regulates osteoclastogenesis. GPR125 may positively regulate osteoclast differentiation through the RANKL-induced MAPK and AKT-NF-κB pathways to regulate the osteoclastogenesis-related transcriptional process and marker gene expression.

Article Snippet: The GPR125 polyclonal antibody (PA5-99891) was from Invitrogen.

Techniques: Marker, Expressing