Journal: Cell Research

Article Title: Early estrogen-induced gene 1, a novel RANK signaling component, is essential for osteoclastogenesis

doi: 10.1038/cr.2013.33

Figure Lengend Snippet: Identification of EEIG1 as a novel RANKL-inducible gene. (A) Genechip analysis of EEIG1 mRNA upon RANKL stimulation. (B) RT-PCR analyses of the mRNA levels for EEIG1, Fam102B, NFATc1, TRAP and GAPDH in RANKL-stimulated BMMs. (C) Expression of EEIG1 and NFATc1 proteins in RANKL-stimulated BMMs. GAPDH served as the loading control. (D) Immunofluorescence staining of EEIG1 (green) in osteoclasts. The nuclei (blue) were stained with DAPI. Scale bar, 50 μm. (E) Quantitative real-time PCR analyses to assess the mRNA level of EEIG1 relative to that of β-actin upon stimulation with LPS, estradiol (E2) or RANKL in BMMs. (F) BMMs were stimulated with RANKL, LPS or E2 for the indicated periods. The stimulated cells were lysed and subjected to western blot analyses with antibodies against EEIG1, NFATc1 and tubulin. Tubulin served as the loading control. (G) Effect of NFATc1 on the expression of EEIG1. BMMs infected with retroviruses expressing control vector or constitutively active NFATc1 (NFATc1-CA) were cultured with M-CSF in the absence or presence of RANKL for 2 days, and then western blot analyses were performed with antibodies against EEIG1, NFATc1 and GAPDH. (H) BMMs were incubated with DMSO (Mock) or CsA (5 μM) in the absence or presence of RANKL for 2 days. Total RNAs were extracted and EEIG1 mRNA levels were examined by quantitative real-time PCR. In all the experiments, BMMs cultured in the presence of M-CSF were stimulated with RANKL, LPS or E2 as indicated.

Article Snippet: The cells were fixed and stained using TRAP staining kit (Sigma).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot, Infection, Plasmid Preparation, Cell Culture, Incubation

Journal: Cell Research

Article Title: Early estrogen-induced gene 1, a novel RANK signaling component, is essential for osteoclastogenesis

doi: 10.1038/cr.2013.33

Figure Lengend Snippet: The C-terminal region of RANK spanning amino acids 532-537 is critical for its interaction with EEIG1. (A, C, E) Schematic diagrams of wild-type and mutant constructs of GST-RANK. Numbers shown above or beneath each protein diagram denote amino acid numbers in RANK. Constructs that can interact with EEIG1 are indicated as +. (B, D, F) 293T cells were transfected with FLAG-tagged full-length EEIG1 together with the indicated GST-tagged RANK or its mutant constructs. The GST-RANK proteins were precipitated with Glutathione-Sepharose beads, and the associated EEIG1 was detected by anti-FLAG antibody. An aliquot of the lysates was reserved to blot the total EEIG1. The GST-RANK expression was detected by Coomassie Brilliant Blue staining (CBB) in B or western blot analyses with anti-GST in D and F. (G, H) RAW 264.7 cells that stably express RANK WT or RANK 532-537 Mt were stimulated with anti-FLAG antibody to induce osteoclast formation. (G) Cells were stained for TRAP activity 4 days after stimulation (left panel). Numbers of TRAP+ MNCs (> 5 nuclei) were counted (right panel). Data are presented as mean ± SD and are representative of at least 3 experiments. Scale bar, 200 μm. *P < 0.001. (H) RAW 264.7 cells were cultured with or without an anti-FLAG antibody for 2 days as indicated. Cells were then lysed and subjected to western blot analyses with antibodies against EEIG1, NFATc1, RANK or GAPDH. GAPDH served as the loading control.

Article Snippet: The cells were fixed and stained using TRAP staining kit (Sigma).

Techniques: Mutagenesis, Construct, Transfection, Expressing, Staining, Western Blot, Stable Transfection, Activity Assay, Cell Culture

Journal: Cell Research

Article Title: Early estrogen-induced gene 1, a novel RANK signaling component, is essential for osteoclastogenesis

doi: 10.1038/cr.2013.33

Figure Lengend Snippet: EIP inhibits osteoclast formation and RANKL-induced bone destruction. (A) Structure of EIP. (B) In vitro differentiation of osteoclasts from BMMs treated with increasing concentrations of EIP. TRAP+ MNCs (>5 nuclei) were counted 4 days after RANKL stimulation. Left panel, TRAP staining; right panel, numbers of TRAP+ MNCs. Data represent mean ± SD. Scale bar, 200 μm. (C) BMMs were stimulated with RANKL in the absence or presence of EIP (10 μM). Cells were lysed and analyzed by western blotting with antibodies against EEIG1, NFATc1, phospho-PLCγ2 and PLCγ2. GAPDH served as the loading control. (D-G) EIP suppresses RANKL-induced (2 mg RANKL/kg body weight) osteoclast formation and bone destruction. TRAP staining was carried out on the sections of mouse calvaria bone (D). Bone morphometric analyses indicate that RANKL-induced increases in the number of osteoclasts (E), the eroded surface (F), and the marrow cavity area (G) were significantly suppressed by the local administration of EIP. Data represent mean ± SD (n = 5). *P <0.01, **P < 0.001.

Article Snippet: The cells were fixed and stained using TRAP staining kit (Sigma).

Techniques: In Vitro, Staining, Western Blot

Journal: Bone Research

Article Title: PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

doi: 10.1038/s41413-022-00201-4

Figure Lengend Snippet: Expression of EP2 and EP4 in subchondral bone osteoclasts and the effects of PGE2 on osteoclasts with EP2 and EP4 deletion. a qRT-PCR analysis of the mRNA expression levels of EP1 , EP2 , EP3 , EP4 , and Trap in osteoclasts differentiated from BMMs exposed to 10 ng·mL −1 M-CSF and 50 ng·mL −1 RANKL for 5–6 days. Error bars are the mean ± s.d. n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001, ns, not significant by unpaired two-tailed Student’s t test. b Representative images of the protein levels of EP2 and EP4 in mouse tibial subchondral bone tissue 2 weeks after sham or ACLT surgery. The experiments were performed with three biological replicates. c Representative images of IHF staining of EP2 or EP4 (green) and TRAP (red) in subchondral bone 2 weeks post-sham or ACLT surgery (left) and quantitative analysis (right). The white arrows indicate TRAP-positive osteoclasts expressing EP2 or EP4. Error bars are the mean ± s.d. * P < 0.05, ns, not significant by unpaired two-tailed Student’s t test. n = 4 for per group. Scale bars, 20 μm. d EP2 deletion inhibits PGE2-induced migration and osteoclast differentiation. BMMs from the EP2 WT and littermate EP2 KO mice were used to generate osteoclasts by stimulation with 10 ng·mL −1 M-CSF and 50 ng·mL −1 RANKL and incubation with 100 nmol·L −1 PGE2. Representative image of cells from the Transwell migration assay (Transwell) and osteoclast differentiation assay (TRAP staining) (top) and the corresponding quantitative analysis (bottom). Error bars are the mean ± s.d. n = 3. Two-way ANOVA followed by Tukey’s t tests. Scale bars, 50 μm. e EP2 deletion inhibits PGE2-induced migration and osteoclast differentiation. BMMs from the WT ( EP4 fl/fl ) and littermate EP4 fl/fl : LysM-cre ( EP4 LysM ) mice were used to generate osteoclasts by stimulation with 10 ng·mL −1 M-CSF and 50 ng·mL −1 RANKL (5–6 days) and incubation with 100 nmol·L −1 PGE2. Representative image of cells from the Transwell migration assay (Transwell) and osteoclast differentiation assay (TRAP staining) (top) and the corresponding quantitative analysis (bottom). Error bars are the mean ± s.d. n = 3. Two-way ANOVA followed by Tukey’s t tests. Scale bars, 50 μm

Article Snippet: When BMMs differentiated into mature osteoclasts, a TRAP staining kit (TaKaRa) was used to stain mature osteoclasts according to the manufacturer′s instructions, and TRAP-positive osteoclasts with 5 or more nuclei were counted.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Staining, Migration, Incubation, Transwell Migration Assay, Osteoclast differentiation Assay

Journal: Bone Research

Article Title: PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

doi: 10.1038/s41413-022-00201-4

Figure Lengend Snippet: EP4 deletion in osteoclasts inhibits OA progression in a murine OA model. a Representative images of TRAP staining and IHF staining of Osterix (Osx), CD115 (red), and RANK (green) in the subchondral bone of the EP4 fl/fl or littermate EP4 LysM mice post-ACLT surgery (top) and quantitative analysis (bottom). TRAP staining, IHF staining of CD115 and RANK, and IHC staining of Osx were performed 2 weeks after ACLT surgery. The white arrow indicates CD115 and RANK double-positive osteoclast precursors. Error bars are the mean ± s.d. Two-way ANOVA followed by Tukey’s t tests. n = 6 for TRAP staining, n = 3 for CD115 and RANK IHF staining. Scale bars, 50 μm (TRAP staining), 20 μm (CD115 and RANK IHF staining), and 20 μm (Osx IHF staining). b Representative 3D reconstructed microCT images, H&E staining, and safranin O-fast green (S.O.) staining of sagittal sections of articular cartilage of the EP4 fl/fl or littermate EP4 LysM mice 8 weeks after ACLT surgery (left). Quantitative analysis of structural parameters of subchondral bone, including Tb.Pf, Tb.Sp and BV/TV. OARSI scores based on the S.O. staining histology analysis (right). Error bars are the mean ± s.d. n = 6 for each group. Two-way ANOVA followed by Tukey’s t tests. Scale bars, 1 mm (microCT), 50 μm (S.O.) and 50 μm (H&E)

Article Snippet: When BMMs differentiated into mature osteoclasts, a TRAP staining kit (TaKaRa) was used to stain mature osteoclasts according to the manufacturer′s instructions, and TRAP-positive osteoclasts with 5 or more nuclei were counted.

Techniques: Staining, Immunohistochemistry

Journal: Bone Research

Article Title: PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

doi: 10.1038/s41413-022-00201-4

Figure Lengend Snippet: The EP4 antagonist HL-43 inhibits OA progression in a murine OA model. a HL-43 inhibits PGE2-induced osteoclast differentiation. BMMs from the EP4 fl/fl mice and littermate EP4 fl/fl : LysM-cre mice ( EP4 LysM ) were used to generate osteoclasts by stimulation with 10 ng·mL −1 M-CSF and 50 ng·mL −1 RANKL and incubation with 100 nmol·L −1 PGE2 in the presence and absence of HL-43 (10 μmol·L −1 ). Representative images of TRAP staining are on the left, and the corresponding quantification is on the right. Error bars are the mean ± s.d. n = 3. Two-way ANOVA followed by Tukey’s t tests. Scale bars, 50 μm. b Representative images of TRAP staining and IHF staining of TRAP (red), Osx (red), CD115 (red), and RANK (green) in the subchondral bone of the WT mice orally treated with the FDA-approved OA pain drug celecoxib (30 mg·kg −1 ) or HL-43 (30 mg·kg −1 ) 2 weeks after ACLT surgery (left) and quantitative analysis (right). The white arrows indicate CD115 and RANK double-positive osteoclast precursors. Error bars are the mean ± s.d. * P < 0.05, ** P < 0. 01 and *** P < 0.001 by one-way ANOVA followed by Tukey’s t tests. n = 4 for TRAP staining, n = 3 for TRAP IHF staining, n = 3 for CD115 and RANK IHF staining, n = 3 for Osx IHC staining. Scale bars, 50 μm (TRAP staining), 20 μm (TRAP IHF staining), 20 μm (CD115 and RANK IHF staining) and 20 μm (Osx IHF staining). c Representative 3D reconstructed microCT images, H&E staining, and S.O. staining of articular cartilage of the WT mice treated with celecoxib or HL-43 8 weeks after ACLT surgery (left). Mice were orally treated daily with celecoxib (30 mg·kg −1 ) or HL-43 (30 mg·kg −1 ) for 8 weeks. Quantitative analysis of structural parameters of subchondral bone, including Tb.Pf, Tb.Sp, and BV/TV. OARSI scores based on S.O. staining histology analysis (right). Error bars are the mean ± s.d. * P < 0.05, ** P < 0.01 and *** P < 0.001, ns, not significant by one-way ANOVA followed by Tukey’s t tests. n = 17 for the sham group, n = 21 for the vehicle-treated group, n = 18 for the celecoxib-treated group, n = 18 for the HL-43-treated group. Scale bars, 1 mm (microCT), 50 μm (H&E) and 50 μm (S.O.)

Article Snippet: When BMMs differentiated into mature osteoclasts, a TRAP staining kit (TaKaRa) was used to stain mature osteoclasts according to the manufacturer′s instructions, and TRAP-positive osteoclasts with 5 or more nuclei were counted.

Techniques: Incubation, Staining, Immunohistochemistry

Journal: Bone Research

Article Title: PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

doi: 10.1038/s41413-022-00201-4

Figure Lengend Snippet: PGE2 regulates migration and osteoclast differentiation through the Gαs/PI3K/AKT/MAPK signaling pathways downstream of EP4. PGE2 regulates migration and osteoclast differentiation independent of β-arrestin1 ( a ) and β-arrestin2 ( b ) but via Gαs ( c ). BMMs from the WT and littermate β-arrestin1- and β-arrestin2- knockout mice were used to generate osteoclasts by stimulation with 10 ng·mL −1 M-CSF and 50 ng·mL −1 RANKL and incubation with 100 nmol·L −1 PGE2. For Gαs experiments, osteoclasts were generated from the GMMs of the Gαs fl/fl controls and Gαs fl/fl ; LysM-cre ( Gαs LysM ) mice. A differentiation assay (TRAP staining) (left) and the corresponding quantitative analysis (right). Scale bars, 50 μm. Error bars are the mean ± s.d. n = 3. Two-way ANOVA followed by Tukey’s t tests. Scale bars, 50 μm. d cAMP production was measured via ELISAs for osteoclasts derived from the Ep4 fl/fl and Ep4 LysM mice. BMMs from the two mouse strains were isolated and stimulated with 10 ng·mL −1 M-CSF and 50 ng·mL −1 RANKL to differentiate into osteoclasts and incubated with 100 nmol·L −1 PGE2 for 30 min prior to cAMP measurements. Error bars are the mean ± s.d. *** P < 0.001 by unpaired two-tailed Student’s t test. The experiment was performed with three biological replicates. e Representative images of the indicated protein expression by western blotting for osteoclasts generated using BMMs of Ep4 fl/fl and Ep4 LysM mice. The cells were treated either with osteoclastogenic media (10 ng·mL −1 M-CSF and 50 ng·mL −1 RANKL) alone, with PGE2 (100 nmol·L −1 ), or PGE2 with IBMX (1 mmol·L −1 ) for 3 h. The experiments were performed with three biological replicates

Article Snippet: When BMMs differentiated into mature osteoclasts, a TRAP staining kit (TaKaRa) was used to stain mature osteoclasts according to the manufacturer′s instructions, and TRAP-positive osteoclasts with 5 or more nuclei were counted.

Techniques: Migration, Knock-Out, Incubation, Generated, Differentiation Assay, Staining, Derivative Assay, Isolation, Two Tailed Test, Expressing, Western Blot

Journal:

Article Title: Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1

doi: 10.1016/j.bone.2006.08.004

Figure Lengend Snippet: Anti-ICAM-1 inhibits ECF-L-induced OCL formation. (A) TRACP staining was performed and the number of TRACP+ multinucleated cells in each well was counted. MDBM cells were treated with RANKL 50 ng/ml (a), RANKL 10 ng/ml (b) and RANKL 10 ng/ml+ECF-L-Fc 200 ng/ml (c) and in the presence of the blocking anti-ICAM 1 antibody RANKL 50 ng/ml, (d) RANKL 10 ng/ml (e) and RANKL 10 ng/ml+ECF-Fc 200 ng/ml (f). (B) Anti-ICAM-1 inhibits ECF-L-induced OCL formation. Anti-ICAM-1 antibody was added to mouse bone marrow cultures stimulated with 200 ng/ml of ECF-L-Fc. Control cultures were treated with a control Fc protein (200 ng/ml) and control IgG protein. Anti-ICAM-1 antibody did not inhibit TRACP+ multinucleated cells induced by 50 ng/ml RANKL, but inhibited TRACP+ MNC formation induced by 10 ng/ml RANKL and 200 ng/ml of ECF-L-Fc. Results represent the mean±SD. A similar pattern of results was seen in three independent experiments. Stippled bars represent cultures treated with control IgG and solid bars are cultures treated with anti-ICAM-1 antibody.

Article Snippet: At the end of culture period, the cells were fixed and stained for tartrate resistant acid phosphatase (TRACP), using a TRACP staining kit (A-367, Sigma) to identify OCL-like multinucleated cells.

Techniques: Staining, Blocking Assay

Journal:

Article Title: Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1

doi: 10.1016/j.bone.2006.08.004

Figure Lengend Snippet: Anti-ICAM-1 inhibits ECF-L-induced OCL formation, treated by 10−10 M 1,25-(OH)2D3 and/or ECF-L-Fc (200 ng/ml). Anti-ICAM-1 antibody was added to mouse bone marrow cultures stimulated with 200 ng/ml of ECF-L-Fc and/or 10−10 M 1,25-(OH)2D3. Control cultures were treated with a control Fc protein (200 ng/ml) and control IgG protein. Anti-ICAM-1 antibody did not inhibit TRACP+ MNC induced by 10−10 M 1,25-(OH)2D3, but inhibited TRACP+ MNC formation induced by 10−10 M 1,25-(OH)2D3 and ECF-L-Fc. Results represent the mean±SD. A similar pattern of results was seen in three independent experiments. Open bars represent cultures not treated with ECF-L and solid bars represent cultures treated with ECF-L.

Article Snippet: At the end of culture period, the cells were fixed and stained for tartrate resistant acid phosphatase (TRACP), using a TRACP staining kit (A-367, Sigma) to identify OCL-like multinucleated cells.

Techniques: