Fig 1: NOG is a target gene of METTL3-mediated m6A modification. A The top terms identified by Gene Ontology enrichment analysis of overlapping DEGs in METTL3-knockdown OM-DPSCs after induction for both 7 and 14 days. B Scatter plots of the top items in Gene Ontology enrichment. C The enrichment analysis of KEGG pathways. D The differentially m6A methylated and expressed genes in the m6A-mRNA profile after induction for 7 and 14 days by m6A RIP & RNA sequencing. E Venn diagram showing overlapping genes between DEGs in transcriptome sequencing after METTL3 knockdown and m6A-methylated transcripts during DPSC mineralization. DEGs: differentially expressed genes; D(m6A)MGs: differentially m6A methylated genes
Fig 2: METTL3 programs NOG destabilization via poly(A) tail shortening. A The remaining NOG mRNA in differentiated and undifferentiated DPSCs treated with actinomycin D was quantified by qPCR. B Protein translation was blocked by cycloheximide, and the remaining expression level of NOG was detected by western blotting (n = 3). C 3'-RACE characterization of the polyadenylation site and 3' UTR information of NOG. D Diagram of 3'-RACE analysis of NOG mRNA. E The poly(A) tail length of the NOG transcript in METTL3 knockdown DPSCs was assayed by poly(A) tail length measurement. F Schematic of the m6A modification machinery in DPSC mineralization. Significance was determined via ANOVA or Student’s t test; the data are represented as the mean ± SD (n = 3). *p < 0.05. **p < 0.01. ***p < 0.001
Fig 3: Stage-specific NOG expression is regulated by METTL3. A The relative gene expression and mRNA expression of NOG in differentiated and undifferentiated DPSCs. B The protein expression level of NOG after METTL3 alteration determined by western blotting (n = 3). C Activation of the downstream Smad signaling pathway was evaluated by measuring the protein expression of p-Smad3, p-Smad1/5 and Smad in OM-DPSCs. D ALP activity and calcium mineralization formation in METTL3-knockdown DPSCs treated with NOG blocking antibody (n = 3). E The RUNX2 and DSPP mRNA expression levels assayed by qPCR. Significance was determined via ANOVA or Student’s t test; the data are presented as the mean ± SD (n = 3). *p < 0.05. **p < 0.01. ***p < 0.001
Fig 4: Dynamic m6A modification of NOG orchestrates the differentiation stage. A The relative gene expression and mRNA expression of NOG during DPSC differentiation. B The protein expression level of NOG detected by western blotting. C Visual data of high-confidence m6A peaks enriched in NOG mRNA detected by m6A RIP-seq. D Details of vectors containing fragments of the NOG 3' UTR with the wild-type m6A motif or two independent m6A mutants (A-to-T mutation). The numbers (450, 463 and 571, 583) represent the positions of the m6A sites relative to the 3' UTR. E qPCR assay of the mRNA expression of NOG in 293T cells transduced with the wild-type or m6A-mutant vector (n = 3). F m6A RIP-qPCR demonstrated the m6A deposition alteration in NOG mRNA after METTL3 inhibition. G Immunofluorescence staining showed the METTL3 and NOG expression in the composites of ß-TCP/HA scaffolds with METTL3-knockdown DPSCs after subcutaneous transplantation in nude mice for 4 weeks. H Subcutaneous transplantation of DPSCs transduced with METTL3 overexpression and control lentivirus. I The expression level of METTL3 and NOG evaluated by imageJ. Significance was determined via ANOVA or Student’s t test; the data are presented as the mean ± SD (n = 3). *p < 0.05. **p < 0.01. ***p < 0.001
Supplier Page from Abcam for Human Noggin peptide