recombinant murine receptor activator of nuclear factor kappa b (nf-κb) ligand (rankl)  (PeproTech)

Journal: Journal of Orthopaedic Translation

Article Title: MAGI1 attenuates osteoarthritis by regulating osteoclast fusion in subchondral bone through the RhoA-ROCK1 signaling pathway

doi: 10.1016/j.jot.2025.04.007

Figure Lengend Snippet: MAGI1 expression in osteoclasts is correlated with the progression of OA. (A, B) RT-qPCR analysis of the expression of osteoclasts marker genes and MAGI family genes in BMDMs during RANKL-induced osteoclastogenesis at 0, 2 and 4 d n = 3. (C) TRAP staining of osteoclasts after M-CSF and RANKL induction at 0, 2 and 4 d. Scale bar, 200 μm. (D) Immunofluorescence co-staining of MAGI1 (green) and F-actin (red) at 0, 2 and 4 days after RANKL induction. Scale bar, 100 μm; 50 μm (magnification). (E) SO-FG staining of the subchondral bone in the OA and RN parts from human osteoarthritic tibial plateau samples. RN = relatively normal part; OA = damaged osteoarthritic part. Scale bar, 1000 μm. (F) Immunofluorescence co-staining of MAGI1 and TRAP in subchondral bone of the OA and RN parts. Scale bar, 200 μm. (G) Quantitative analysis of MAGI1 intensity in subchondral bone of the OA and RN parts. n = 5. (H) SO-FG and TRAP staining to assess cartilage and subchondral bone damage in mice at 4 weeks after DMM surgery. Scale bar, 200 μm (upper), 100 μm (middle), 100 μm (bottom). (I) Immunofluorescence co-staining of MAGI1 (green) and TRAP (red) in tibial subchondral bone marrow at 4 weeks after DMM surgery. Scale bar, 200 μm. (J, K) Statistics of ratio of hyaline cartilage to calcified cartilage thickness, and quantification of TRAP + osteoclasts in subchondral bone marrow. n = 5. (L) Quantitative analysis of MAGI1 fluorescence intensity in subchondral bone marrow, n = 4. Error bars are means ± s.d., ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, by one-way ANOVA with several comparisons employing the Tukey method. Comparisons between two groups were performed using the two-tailed Student’s t- test.

Article Snippet: Recombinant murine macrophage colony-stimulating factor (M-CSF) and recombinant murine RANKL were acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Marker, Staining, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: Journal of Orthopaedic Translation

Article Title: MAGI1 attenuates osteoarthritis by regulating osteoclast fusion in subchondral bone through the RhoA-ROCK1 signaling pathway

doi: 10.1016/j.jot.2025.04.007

Figure Lengend Snippet: Overexpression of MAGI1 in preosteoclasts suppresses osteoclastogenesis in vitro. (A) TRAP staining of osteoclasts derived from RANKL-treated BMDMs cells with overexpression of MAGI1 on 5–6 d. Scale bar, 200 μm. (B) Quantitative analysis of TRAP-positive cell number in per well. n = 3. (C) Actin ring structures of osteoclasts derived from RANKL-treated BMDMs with overexpression of MAGI1 on 5–6 d. Scale bar, 200 μm. (D) Quantitative analysis of actin rings in per well. n = 3. (E) RT-qPCR detected the osteoclast-related gene expression levels of Acp5, Nfatc1, c-Fos, Atp6v0d2, Dc-stamp, and Ctsk in the NC and OE-MAGI1 groups on 4 d n = 3. Error bars are means ± s.d., ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, by one-way ANOVA with several comparisons employing the Tukey method. Comparisons between two groups were performed using the two-tailed Student’s t -test.

Article Snippet: Recombinant murine macrophage colony-stimulating factor (M-CSF) and recombinant murine RANKL were acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Over Expression, In Vitro, Staining, Derivative Assay, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Journal of Orthopaedic Translation

Article Title: MAGI1 attenuates osteoarthritis by regulating osteoclast fusion in subchondral bone through the RhoA-ROCK1 signaling pathway

doi: 10.1016/j.jot.2025.04.007

Figure Lengend Snippet: Targeting MAGI1-RhoA-ROCK1 pathway regulate osteoclastogenesis. (A) RT-qPCR analysis of RhoA and ROCK1 mRNA levels following MAGI1 overexpression. n = 3. (B) Western blotting was used to detect the protein levels of RhoA and ROCK1. GAPDH served as the internal control. (C) Western blotting analysis for p-p65, p65, and c-Fos in the RANKL-treated BMDMs. GAPDH served as the internal control. (D) RT-qPCR analysis of the effect of Narc on ROCK1, c-Fos, and Ctsk after overexpression of MAGI1. n = 3. (E, F) Immunofluorescence staining of F-actin (red), ROCK1 (green), and nucleus (blue) with addition of Narc, and quantification of ROCK1 fluorescence. Scale bar, 25 μm. n = 3. (G) Immunofluorescence staining of p-p65 with Narc and without Narc. Scale bar, 25 μm (Left), 10 μm (Right). (H) Statistical analysis of relative fluorescence intensity of p-p65. n = 3. (I) TRAP staining was performed to detect the differentiation of osteoclasts with or without the Narc. Scale bar, 200 μm. (J) Quantitative analysis of TRAP-positive cell numbers in per well. n = 3. Error bars are means ± s.d., ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, by one-way ANOVA with multiple comparisons using the Tukey method. Comparisons between two groups were performed using the two-tailed Student’s t -test.

Article Snippet: Recombinant murine macrophage colony-stimulating factor (M-CSF) and recombinant murine RANKL were acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Quantitative RT-PCR, Over Expression, Western Blot, Control, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: Journal of Orthopaedic Translation

Article Title: MAGI1 attenuates osteoarthritis by regulating osteoclast fusion in subchondral bone through the RhoA-ROCK1 signaling pathway

doi: 10.1016/j.jot.2025.04.007

Figure Lengend Snippet: Schematic model of mechanisms. During OA onset, overactivation of osteoclasts leads to subchondral bone remodeling, increased angiogenesis and sensory innervation invasion. During RANKL-induced osteoclast differentiation, high expression of MAGI1 in osteoclasts suppressed the RhoA/ROCK1 signaling pathway, which subsequently inhibited NF-κB-mediated osteoclastogenesis (Scheme created with BioRender).

Article Snippet: Recombinant murine macrophage colony-stimulating factor (M-CSF) and recombinant murine RANKL were acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing