Journal: Journal of Orthopaedic Translation

Article Title: Magnesium ions facilitate osteogenic differentiation and intervertebral fusion via m6A methylation of RhoA mRNA

doi: 10.1016/j.jot.2026.101056

Figure Lengend Snippet: Magnesium ions enhance osteogenesis through upregulating METTL3. (A) Dot blot experiments were conducted on MC3T3-E1 cells from different groups to determine the mRNA methylation levels and gray-scale analysis was performed. (B) The content of m6A modification in total RNA of MC3T3-E1 cells in different groups was determined by using the m6A RNA methylation quantification kit. (C-D) RT-qPCR was employed to assess the alterations in the mRNA levels of METTL3, ALKBH5, METTL14 and FTO within MC3T3-E1 cells following 4 mmol/L magnesium treatment. (E) The content of m6A modification in total RNA of MC3T3-E1 cells in different groups was determined by using the m6A RNA methylation quantification kit. (F) RT-qPCR was employed to assess the alterations in the mRNA levels of METTL3, Runx2 and OCN within MC3T3-E1 cells following different treatments. (G-H) Western blotting was utilized to measure the protein levels of METTL3, Runx2, and OCN in cells from different treatment groups. (I) MC3T3-E1 cells were subjected to ALP staining and ARS staining, and the results were subsequently quantified. (Data are expressed as mean ± SD (n = 3). Statistical significance was determined by Student's t-test (between two groups) or one-way ANOVA (among multiple groups)).

Article Snippet: To inhibit METTL3's capacity to promote m6A modification, cells were treated with 10 μmol/L of the METTL3 functional inhibitor STM2457 (HY-134836, MedChemExpress) for 24 h [ ].

Techniques: Dot Blot, Methylation, Modification, Quantitative RT-PCR, Western Blot, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Magnesium ions facilitate osteogenic differentiation and intervertebral fusion via m6A methylation of RhoA mRNA

doi: 10.1016/j.jot.2026.101056

Figure Lengend Snippet: Knocking down RhoA inhibits osteogenesis. (A) Western blotting was employed to assess the expression levels of METTL3, RhoA, and ROCK1 proteins in cells from different groups. (B) RT-qPCR was employed to assess the expression levels of METTL3, RhoA, and ROCK1 mRNA in cells from different groups. (C) Small interfering RNAs (siRNAs) were utilized to knockdown RhoA, and the knockdown efficiency was validated via Western blotting. (D) RT-qPCR was employed to assess the expression levels of METTL3, RhoA, Runx2, OCN and ROCK1 mRNA in cells from different groups. (E) Western blotting was employed to assess the expression levels of METTL3, Runx2, OCN and ROCK1 proteins in cells from different groups. (F) MC3T3-E1 cells were subjected to ALP staining and ARS staining, and the results were subsequently quantified. (Data are presented as mean ± SD from three independent experiments (n = 3). Statistical differences were analyzed using one-way ANOVA. Post-hoc pairwise comparisons were conducted using the LSD test.).

Article Snippet: To inhibit METTL3's capacity to promote m6A modification, cells were treated with 10 μmol/L of the METTL3 functional inhibitor STM2457 (HY-134836, MedChemExpress) for 24 h [ ].

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Knockdown, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Magnesium ions facilitate osteogenic differentiation and intervertebral fusion via m6A methylation of RhoA mRNA

doi: 10.1016/j.jot.2026.101056

Figure Lengend Snippet: Magnesium facilitates the fusion of rat caudal vertebrae via the regulation of METTL3 and RhoA. (A) Flowchart of animal experiments. (B) Magnesium ion quantification assays were performed on newly formed bone tissues from different groups using a magnesium ion quantitative kit. (C) Determination of magnesium ion concentration in tissues at different time points after surgery. Based on the in vitro experimental findings of this study, the effective concentration range is 2–6 mmol, with the safe concentration range being <16 mmol. (D) MeRIP-qPCR was used to detect the m6A modification abundance of RhoA mRNA in the newly formed bone tissue. (E) qPCR experiments were conducted on the new bone tissues of different groups to determine the expression levels of different mRNAs. (F) Western Blotting experiments were conducted on bone tissues of different groups to determine the expression levels of different proteins. (Data are presented as mean ± SD from three independent experiments (n = 3). Statistical differences were analyzed using one-way ANOVA. Post-hoc pairwise comparisons were conducted using the LSD test.).

Article Snippet: To inhibit METTL3's capacity to promote m6A modification, cells were treated with 10 μmol/L of the METTL3 functional inhibitor STM2457 (HY-134836, MedChemExpress) for 24 h [ ].

Techniques: Concentration Assay, In Vitro, Modification, Expressing, Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: Magnesium ions facilitate osteogenic differentiation and intervertebral fusion via m6A methylation of RhoA mRNA

doi: 10.1016/j.jot.2026.101056

Figure Lengend Snippet: Imaging and histological detection of intervertebral fusion of the tail vertebrae in rats. (A) Micro-CT scan images of bones at different times in different groups. (B) H&E-stained sections of bone tissues from each group at 2 weeks post-surgery. (C) Goldner - stained sections of bone tissues from each group at 2 weeks post - operation. (D) Masson - stained sections of bone tissues from each group at 2 weeks post - operation. (E) Immunofluorescent staining sections of bone tissues from each group at 2 weeks post - surgery (DAPI stained blue, RhoA stained red, METTL3 stained yellow, and Runx2 stained green).

Article Snippet: To inhibit METTL3's capacity to promote m6A modification, cells were treated with 10 μmol/L of the METTL3 functional inhibitor STM2457 (HY-134836, MedChemExpress) for 24 h [ ].

Techniques: Imaging, Micro-CT, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Magnesium ions facilitate osteogenic differentiation and intervertebral fusion via m6A methylation of RhoA mRNA

doi: 10.1016/j.jot.2026.101056

Figure Lengend Snippet: Magnesium ions upregulate METTL3 expression, enhancing m6A modification on RhoA mRNA. The m6A reader YTHDF1 recognizes and binds to the modified sites, promoting RhoA translation. This activates the RhoA/ROCK signaling pathway, ultimately driving osteogenic differentiation, bone remodeling, and intervertebral fusion.

Article Snippet: To inhibit METTL3's capacity to promote m6A modification, cells were treated with 10 μmol/L of the METTL3 functional inhibitor STM2457 (HY-134836, MedChemExpress) for 24 h [ ].

Techniques: Expressing, Modification