Journal: Experimental & Molecular Medicine

Article Title: METTL3-mediated pre-miR-665/DLX3 m 6 A methylation facilitates the committed differentiation of stem cells from apical papilla

doi: 10.1038/s12276-024-01245-8

Figure Lengend Snippet: a Flowchart of the m 6 A-RIP microarray analysis procedure used to identify the targets of METTL3. b The top 5 pre-miRNAs with upregulated METTL3-mediated m 6 A methylation are listed. c The combined sites of pre-miR-665 and m 6 A methylation are labeled. d MeRIP-qPCR showed the positive effect of METTL3-OE on pre-miR-665 methylation by m 6 A compared with that of NC-OE. e qRT‒PCR analysis of the inhibitory impact of pre-miR-665 and miR-665 on METTL3 overexpression. f MeRIP-qPCR showed the negative effect of si-METTL3 on pre-miR-665 methylation by m 6 A compared with that of si-NC. g qRT‒PCR analysis of the ability of pre-miR-665 and miR-665 to promote METTL3 inhibition. h The mRNA expression of DSPP, DMP1, ALP, RUNX2 and OSX in the SCAPs transfected with miR-665 mimics and inhibitors. i , j The protein expression of DSPP, DMP1, ALP, RUNX2 and OSX and quantitative analysis in the SCAPs transfected with miR-665 mimics and inhibitors. k – n Representative images of ALP- and ARS-stained transfected SCAPs at 7 and 14 days in mineralized medium. Scale bar = 200 µm. o Immunofluorescence staining results of transfected SCAPs stained for ALP and RUNX2. Scale bar = 40 µm. p qRT‒PCR indicated pre-miR-665 was a target of YTHDF2. q si-YTHDF2 increased the expression of pre-miR-665 after cotransfection of METTL3-OE. n = 3, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

Article Snippet: The cRNAs were combined and hybridized onto the Arraystar Mouse Epitranscriptomic Microarray (8 × 60 K, Arraystar).

Techniques: Microarray, Methylation, Labeling, Over Expression, Inhibition, Expressing, Transfection, Staining, Immunofluorescence, Cotransfection

Journal: Experimental & Molecular Medicine

Article Title: METTL3-mediated pre-miR-665/DLX3 m 6 A methylation facilitates the committed differentiation of stem cells from apical papilla

doi: 10.1038/s12276-024-01245-8

Figure Lengend Snippet: a Potential sites and regions for m 6 A modification in the sequence of the DLX3 gene. b, c Screening for target genes with increased expression and m 6 A methylation levels in SCAPs after overexpression of METTL3 through m 6 A-RIP microarray sequencing. d MeRIP-qPCR illustrated that METTL3 overexpression promoted the m 6 A modification of the DLX3 gene in SCAPs. e – h Western blot analysis confirmed a positive correlation between METTL3 and DLX3 expression. i The half-life (t 1/2 ) of DLX3 mRNA in the SCAPs in the control, METTL3-OE and si-METTL3 groups was measured by qRT‒PCR. j The expression of DLX3 in the SCAPs transfected with control, si-YTHDF1, si-IGF2BP1, si-IGF2BP2 and si-IGF2BP3 was verified by qRT‒PCR. k , l Western blotting results showing that the increase in DLX3 expression caused by METTL3-OE was reversed by si-YTHDF1. m , n si-DLX3 downregulated the expression of odonto/osteogenic protein markers in SCAPs in the presence of METTL3-OE. o ARS staining results illustrated that si-DLX3 slightly reversed the generation of mineralized nodules increased by METTL3-OE in SCAPs. Scale bar = 100 µm. p Immunofluorescence staining showed that compared with WT mice, METTL3 +/− mice exhibited weaker DLX3 expression. Scale bar = 40 µm. q A scheme showing the role of the METTL3/pre-miR-665/DLX3 regulatory network in SCAPs (By Figdraw). n = 3, ns indicates no significance; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

Article Snippet: The cRNAs were combined and hybridized onto the Arraystar Mouse Epitranscriptomic Microarray (8 × 60 K, Arraystar).

Techniques: Modification, Sequencing, Expressing, Methylation, Over Expression, Microarray, Western Blot, Control, Transfection, Staining, Immunofluorescence