n6 methyladenosine m6a  (TargetMol)

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: 600 μg of total RNA was mixed with 12 μg of anti-m6A antibody (ab151230, Abcam) in IP buffer (10 mmol/L pH 7.4 Tris-HCl, 150 mmol/L NaCl, 0.1% NP-40, and 40 U/μL RNAse inhibitor) and incubated at 4 °C with rotation for 2 h. To block, 15 μL of protein A (LSKMAGA10, Millipore) and 15 μL of protein G (LSKMAGG10, Millipore) magnetic beads were added to the IP buffer containing BSA (0.5 mg/mL) and incubated at 4 °C with rotation for 2 h. The IP mixture and blocked magnetic beads were then combined and incubated at 4 °C with rotation for 2 h. The mixture was eluted with m6A 5′-monophosphate sodium salt (6.7 mmol/L, sc-215524, Santa Cruz) dissolved in IP buffer, and the eluates were combined.

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: Mg facilitate osteogenesis through the regulation of RhoA. (A) mRNA methylation microarray assays were conducted on cells from different groups. All probe sites exhibiting differences were meticulously recorded, and the corresponding transcripts were comprehensively analyzed. A heatmap was employed to visually represent all mRNAs with differential expression levels (left). Cluster analysis was then carried out on the assay results, where each data point represented a distinct transcript. The logarithm of the mRNA expression level was designated as the X - axis, and the negative logarithm of the p - value was set as the Y - axis. Using a threshold of p < 0.05 and |Fold Change| > 1.5, a total of 555 genes with significantly upregulated expression and 187 genes with significantly downregulated expression were identified. Select genes are labeled within the figure (right). (B) mRNA methylation microarray analyses were conducted on cells from different groups. All probe sites showing differential signals were precisely recorded, and the associated transcripts were subsequently analyzed. A heatmap was utilized to visualize all mRNAs presenting differences in m6A modification levels (left). Cluster analysis was then performed on the assay outcomes, with each data point representing a distinct transcript. The logarithm of the m6A modification level was designated as the X - axis, while the negative logarithm of the p - value was set as the Y - axis. Using a threshold of p < 0.05 and |Fold Change| > 1.5, a total of 682 genes with significantly upregulated m6A levels and 167 genes with significantly downregulated m6A levels were identified. A selection of these genes is labeled in the figure (right). (C) Select all genes with m6A modification level results showing p < 0.05 (irrespective of the value of Fold Change) and all genes with mRNA expression level results showing p < 0.05 (regardless of the magnitude of Fold Change) that were screened out from the cluster analysis. The intersection of these two gene sets was determined using a Venn diagram, yielding 633 potential genes. (D) Cluster analysis was performed on the potential genes screened from Figure C. Each data point in the analysis represents a distinct transcript. The logarithm of the m6A modification level was designated as the X - axis, while the inter - group difference (Diff) in the percentage of m6A - modified transcripts relative to total mRNA transcripts was set as the Y - axis. Using a threshold of Diff >5% and |Fold Change| > 1.5, a total of 97 genes were identified. These genes exhibit both an up - regulation in the percentage of m6A modification and an increase in the m6A modification level. A number of these genes are labeled in figure. (E) All differentially expressed genes were selected for GO analysis. The enrichment scores of genes under each GO term were calculated through GO analysis. After taking the logarithm, relevant GO classifications with high enrichment scores were sorted and listed in three dimensions: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). (F) For the GO terms related to biological process selected in Figure E, a dot plot was constructed. In this plot, the p - value differences were visually represented by the color of the dots, the number of genes was shown by the dot size, and the enrichment scores were depicted on the X - axis to display potential pathway entries. (G) For the GO terms related to cellular components selected in Figure E, a dot plot was constructed. (H) For the GO terms related to molecular functions selected in Figure E, a dot plot was constructed. (I) The bar chart of KEGG analysis depicts the identified signaling pathways. The enrichment score is presented on the X - axis, the pathway name is shown on the Y - axis, and the length of each bar corresponds to the number of genes associated with that particular pathway. (J) Perform KEGG pathway analysis on genes of statistical significance identified in the clustering analysis. Screen for cellular pathways that contain a large number of genes with differential m6A modification levels and genes with differential mRNA expression levels. On the pathway map, genes with upregulated m6A modification levels are labeled in yellow, genes with upregulated mRNA modification levels only are labeled in red, and active genes that show no significant expression differences but have relatively high intracellular levels are labeled in green. The figure presented herein shows the pathway map of the staining of peripheral genes associated with the selected RhoA - ROCK pathway. (H) Select all the active genes with statistical significance in the cluster analysis, and draw a network relationship diagram of the important pathway genes related to the RhoA gene among them. In this diagram, dots represent genes and lines represent relationships. The size of the dots indicates the significance level, and the thickness of the lines represents the literature support for the interaction.

Article Snippet: 600 μg of total RNA was mixed with 12 μg of anti-m6A antibody (ab151230, Abcam) in IP buffer (10 mmol/L pH 7.4 Tris-HCl, 150 mmol/L NaCl, 0.1% NP-40, and 40 U/μL RNAse inhibitor) and incubated at 4 °C with rotation for 2 h. To block, 15 μL of protein A (LSKMAGA10, Millipore) and 15 μL of protein G (LSKMAGG10, Millipore) magnetic beads were added to the IP buffer containing BSA (0.5 mg/mL) and incubated at 4 °C with rotation for 2 h. The IP mixture and blocked magnetic beads were then combined and incubated at 4 °C with rotation for 2 h. The mixture was eluted with m6A 5′-monophosphate sodium salt (6.7 mmol/L, sc-215524, Santa Cruz) dissolved in IP buffer, and the eluates were combined.

Techniques: Methylation, Microarray, Quantitative Proteomics, Expressing, Labeling, Modification, Selection, Construct, Protein-Protein interactions, 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: YTHDF1 promotes translation of RhoA mRNA upon binding to it. (A) The methylation site sequences and their predicted reliability were screened out based on the probe sequence data of the m6A methylation detection chip through SRAMP analysis. (B) The MeRIP-PCR experiment was conducted to verify the accuracy of the predicted m6A sites. RhoA wild-type (RhoA-WT) and m6A site mutant (RhoA-MUT) primers were used to test the two groups of cells. (C) Utilizing the TFBS (Transcription Factor Binding Site) database, genes capable of binding to the modified sequences at the 3′UTR region were screened. These genes were then correlated with those associated with methylation modification regulation entries in the GO analysis. A Venn diagram was constructed to identify the intersection, thereby screening out potential candidate genes. (D) Following the pulldown assay, protein electrophoresis bands of the positive control group (Input), experimental group (Pulldown), and negative control group (NC) were subjected to Coomassie Brilliant Blue staining. (E) After the pulldown assay, Western Blotting analysis was performed on the proteins from each group to validate their enrichment levels. (F) RIP‐derived protein and RNA in MC3T3-E1 cells examined using Western blotting and RT-qPCR, respectively. (G) After treating MC3T3-E1 cells with actinomycin D and si-YTHDF1, mRNA was detected by qRT-PCR at 0, 3, 6, and 9 h, respectively. The Y-axis represents the relative level of mRNA compared to 0 h. (H) After treating MC3T3-E1 cells with actinomycin D and OE-YTHDF1, mRNA was detected by qRT-PCR at 0, 3, 6 and 9 h, respectively. (I-J) After treating MC3T3-E1 cells with CHX and si-YTHDF1, protein was detected by WB at 0, 3, 6, and 9 h, respectively. The Y-axis represents the relative level of protein compared to 0 h. (K-L) After treating MC3T3-E1 cells with CHX and OE-YTHDF1, protein was detected by WB at 0, 3, 6, and 9 h, respectively. (M) After treating MC3T3-E1 cells with si-YTHDF1, qRT-PCR analysis of RhoA mRNA distribution in different ribosome populations. (N) After treating MC3T3-E1 cells with OE-YTHDF1, qRT-PCR analysis of RhoA mRNA distribution in different ribosome populations. (O) YTHDF1's ability to recognize m6A modifications depends on m6A-binding pockets in the YTH domain. (P) RIP-derived protein and RNA in MC3T3-E1 cells examined using western blotting and RT-qPCR, respectively. (Q) WB was used to detect RhoA expression in the MC3T3-E1 cells. (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: 600 μg of total RNA was mixed with 12 μg of anti-m6A antibody (ab151230, Abcam) in IP buffer (10 mmol/L pH 7.4 Tris-HCl, 150 mmol/L NaCl, 0.1% NP-40, and 40 U/μL RNAse inhibitor) and incubated at 4 °C with rotation for 2 h. To block, 15 μL of protein A (LSKMAGA10, Millipore) and 15 μL of protein G (LSKMAGG10, Millipore) magnetic beads were added to the IP buffer containing BSA (0.5 mg/mL) and incubated at 4 °C with rotation for 2 h. The IP mixture and blocked magnetic beads were then combined and incubated at 4 °C with rotation for 2 h. The mixture was eluted with m6A 5′-monophosphate sodium salt (6.7 mmol/L, sc-215524, Santa Cruz) dissolved in IP buffer, and the eluates were combined.

Techniques: Binding Assay, Methylation, Sequencing, Mutagenesis, Modification, Construct, Protein Electrophoresis, Positive Control, Negative Control, Staining, Western Blot, Derivative Assay, Quantitative RT-PCR, Expressing

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: 600 μg of total RNA was mixed with 12 μg of anti-m6A antibody (ab151230, Abcam) in IP buffer (10 mmol/L pH 7.4 Tris-HCl, 150 mmol/L NaCl, 0.1% NP-40, and 40 U/μL RNAse inhibitor) and incubated at 4 °C with rotation for 2 h. To block, 15 μL of protein A (LSKMAGA10, Millipore) and 15 μL of protein G (LSKMAGG10, Millipore) magnetic beads were added to the IP buffer containing BSA (0.5 mg/mL) and incubated at 4 °C with rotation for 2 h. The IP mixture and blocked magnetic beads were then combined and incubated at 4 °C with rotation for 2 h. The mixture was eluted with m6A 5′-monophosphate sodium salt (6.7 mmol/L, sc-215524, Santa Cruz) dissolved in IP buffer, and the eluates were combined.

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: 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: 600 μg of total RNA was mixed with 12 μg of anti-m6A antibody (ab151230, Abcam) in IP buffer (10 mmol/L pH 7.4 Tris-HCl, 150 mmol/L NaCl, 0.1% NP-40, and 40 U/μL RNAse inhibitor) and incubated at 4 °C with rotation for 2 h. To block, 15 μL of protein A (LSKMAGA10, Millipore) and 15 μL of protein G (LSKMAGG10, Millipore) magnetic beads were added to the IP buffer containing BSA (0.5 mg/mL) and incubated at 4 °C with rotation for 2 h. The IP mixture and blocked magnetic beads were then combined and incubated at 4 °C with rotation for 2 h. The mixture was eluted with m6A 5′-monophosphate sodium salt (6.7 mmol/L, sc-215524, Santa Cruz) dissolved in IP buffer, and the eluates were combined.

Techniques: Expressing, Modification