Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification of pri-miRNA-31 promotes hypertrophic scar progression

doi: 10.3724/abbs.2025033

Figure Lengend Snippet: miR-31-5p counteracts the effects of m 6 A methylation inhibitors on fibrosis and cell proliferation (A) miR-31-5p mimics partially reversed the m6A methylation inhibitor-mediated regulation of COL I/III and α-SMA in HSFBs. (B) Grayscale analysis of COL I/III and α-SMA levels. (C) miR-31-5p mimics partially attenuated the m6A methylation inhibitor-mediated regulation of cell proliferation in HSFBs. (D) Percentage of EdU-positive cells. (E) CCK-8 assay was used to measure the proliferative capacity of the cells from 24 to 96 h. (F) Percentage of EdU-positive cells. Groups: miR-31-5p-negative control (NC); miR-31-5p-NC + cycloleucine (CL, 40 μM) treatment; miR-31-5p-mimics; miR-31-5p-mimics + CL treatment. NS, not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 50 μm.

Article Snippet: To inhibit m 6 A methylation, the cells were exposed to 40 μM cycloleucine (CL, HY-30008; MCE, Monmouth Junction, USA) for 48 h. Additionally, to inhibit METTL3, the cells were treated with 20 μM STM2457 (S9870; Selleck, Huston, USA) or a control (DMSO equivalent) for 24 h.

Techniques: Methylation, CCK-8 Assay, Negative Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification of pri-miRNA-31 promotes hypertrophic scar progression

doi: 10.3724/abbs.2025033

Figure Lengend Snippet: ZBTB20 is a downstream target gene of miR-31-5p (A) Heatmap of genes differentially expressed between the METTL3-knockdown groups and the control groups. (B) Volcano map of genes differentially expressed between the METTL3-knockdown groups and the control groups. (C) Venn diagram illustrating the overlap between the miRDB and TargetScan predictions and upregulated genes in METTL3-knockdown cells. ZBTB20, LITAF and SEPHS1 are downstream target genes of miR-31-5p. (D) Western blot analysis of ZBTB20, LITAF and SEPHS1 protein levels. (E) Grayscale analysis of ZBTB20, LITAF and SEPHS1. (F) The mRNA levels of ZBTB20, LITAF and SEPHS1 were detected by qRT-PCR. Groups: miR-31-5p-negative control (NC); miR-31-5p-NC + cycloleucine (CL, 40 μM) treatment; miR-31-5p-mimics; miR-31-5p-mimics + CL treatment. (G) The METTL3 inhibitor STM2457 can suppress the mRNA levels of ZBTB20 mediated by the miR-31-5p mimic. Groups: miR-31-5p-negative control (NC); miR-31-5p-NC + STM2457 treatment; miR-31-5p-mimics; miR-31-5p-mimics + STM2457 treatment. (H) Information on the mutated sequence of ZBTB20 used for Dual-Luciferase reporter gene assays. (I) Dual-luciferase reporter gene assays confirmed that ZBTB20 is a downstream target gene of miR-31-5p. (J) Knockdown of ZBTB20 increases the mRNA levels of COL I/III and α-SMA. ZBTB20-knockdown groups: si-ZBTB20-1# and si-ZBTB20-2#; control group: negative control (NC). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: To inhibit m 6 A methylation, the cells were exposed to 40 μM cycloleucine (CL, HY-30008; MCE, Monmouth Junction, USA) for 48 h. Additionally, to inhibit METTL3, the cells were treated with 20 μM STM2457 (S9870; Selleck, Huston, USA) or a control (DMSO equivalent) for 24 h.

Techniques: Knockdown, Control, Western Blot, Quantitative RT-PCR, Negative Control, Sequencing, Luciferase

Journal: Therapeutic Advances in Chronic Disease

Article Title: N 6 -methyladenosine demethylases Alkbh5/Fto regulate cerebral ischemia-reperfusion injury

doi: 10.1177/2040622320916024

Figure Lengend Snippet: Betaine treatment enhanced OGD/R treated primary neuronal death and CL treatment attenuated OGD/R-treated primary neuronal death. (A–C) Primary neurons were treated with 8 mM Betaine or 20mM CL and then cultured under OGD/R conditions or normoxia. Cell death was quantified by CCK8 assay or by microscopy for PI-positive cells. Quantifications of the percentage of dead cells were shown. (mean ± SEM; n = 6–20; * p < 0.05, *** p < 0.001 versus OGD/R). (D–E) Representative images of Western blots of primary neuron treated with 8 mM Betaine or 20 mM CL. Cleaved-caspase3 levels were determined by Western blot (mean ± SEM; n = 3; * p < 0.05 versus OGD/R). (F) A proposed model for Alkbh5 and Fto in cerebral ischemia-reperfusion injury. CCK8, Cell Counting Kit-8; CL, cycloleucine; OGD/R, glucose oxygen deprivation/reoxygenation; PI, propidium iodide; SEM, standard error of the mean.

Article Snippet: Neurons were cultured in vitro for neuron standard medium supplemented with cycloleucine (CL) [A1063, TCI (Shanghai) Chemical Industry Development Co., Ltd.] (10 mM, 20 mM, 40 mM) and betaine (61962, Sigma) (4 mM, 8 mM, 16 mM) from 24 h before OGD to the end of the experiment.

Techniques: Cell Culture, CCK-8 Assay, Microscopy, Western Blot, Cell Counting

Journal: International Journal of Oral Science

Article Title: METTL7A-mediated m6A modification of corin reverses bisphosphonates-impaired osteogenic differentiation of orofacial BMSCs

doi: 10.1038/s41368-024-00303-1

Figure Lengend Snippet: METTL7A promoted corin m6A methylation level. a , b Treated with 500 µg/mL Cycloleucine, representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-CORIN Group. c Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/oe-CORIN Group. d , e qRT-PCR analyses, as well as representative immunoblotting show corin expression from sh-NC/sh-METTL7A Group. f MeRIP-qPCR results show m6A modification of corin mRNA from sh-NC/sh-METTL7A Group. g Comparation of corin mRNA stability from sh-NC/sh-METTL7A Group. h , i Corin expression from Vector/oe-METTL7A Group. j MeRIP-qPCR results show m6A modification on corin mRNA from Vector/oe-METTL7A Group. k Comparation of corin mRNA stability from Vector/oe-METTL7A Group. l Construct of Wildtype and mutant 3’UTR CORIN plasmids. m METTL7A binding to the CORIN 3′UTR is verified by the dual-luciferase reporter assay. Data are expressed as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, sh-NC or Vector vs. sh-METTL7A or oe-METTL7A. ### P < 0.001, oe-CORIN vs oe-CORIN+Cycloleucine

Article Snippet: We found that ALP/ARS staining and Runx2/OCN expression in the Cycloleucine treated oe-CORIN Group were much lower than the untreated oe-CORIN Group (Fig. ), suggesting that m6A modification of corin influenced osteogenic differentiation.

Techniques: Methylation, Staining, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Modification, Construct, Mutagenesis, Binding Assay, Luciferase, Reporter Assay

Journal: Neurobiology of Stress

Article Title: Erasing m 6 A-dependent transcription signature of stress-sensitive genes triggers antidepressant actions

doi: 10.1016/j.ynstr.2021.100390

Figure Lengend Snippet: TCAs act by erasing m 6 A modification via an FTO-dependent manner. (A, top) Schematic timeline of cycloleucine injection and behavioral tests. Bottom, reducing m 6 A RNA methylation in the VTA by microinjection of cycloleucine (40 μg/μL/side) decreased the immobility time of TST and FST in mice (n = 8–10 mice/group, Student's t -test). (B, top) Schematic timeline of CSDS, cycloleucine treatment and behavior tests. Animals were subjected to SIT and SPT before the cannulation to identify the susceptible group, and only the mice with both interaction ratio <1.0 in SIT and sucrose preference <75% in SPT were considered to be susceptible and given vehicle or cycloleucine. Bottom , cycloleucine increases social interaction ratio and sucrose preference in susceptible mice (n = 7–9 mice/group, two-way ANOVA, Bonferroni's test). ( C) Intraperitoneal delivery of rhein (120 mg/kg) blocks the effect of IMI and AMT on immobility time of FST and TST (n = 11–12 mice/group, one-way ANOVA, Bonferroni's test). (D) Local bilateral infusion with rhein (1 μg/μL, 1μL/side) eliminated the antidepressant effect of IMI and AMT (n = 5–10 mice/group, two-way ANOVA, Bonferroni's test). (E) Genetically knockdown of FTO in the VTA abolished the effect of IMI on immobility time of TST and FST in mice (n = 5–7 mice/group, two-way ANOVA, Bonferroni's test). ( F ) IMI reverses the decrease in social interaction and sucrose preference of stressed mice, while FTO knockdown blocked the therapeutic effect of IMI (n = 8 mice/group, one-way ANOVA, Bonferroni's test). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01. FST, forced swim test; TST, tail suspension test; SPT, sucrose preference test; SIT, social interaction test.

Article Snippet: Cycloleucine (40 μg/μL/side, Sinopharm, Shanghai, China), betaine (20 μg/μL/side, Sinopharm, Shanghai, China), rhein (1 μg/μL/side, Aladdin, Shanghai, China), recombinant urocortin (1 μg/μL/side, Cloud-Clone Crop, Wuhan, China) and recombinant cocaine and amphetamine regulated transcript (CART, 0.5 μg/μL/side, Immuno Clone Biosciences Co., Ltd, Huston, USA) were microinjected into the VTA through the cannulas with the microsyringe pump.

Techniques: Modification, Injection, Methylation, Microinjection, Knockdown, Suspension