Journal: Acta biochimica et biophysica Sinica

Article Title: Melatonin mitigates ovarian aging through regulation of the YTHDF2/m 6 A/UBE3C axis.

doi: 10.3724/abbs.2025090

Figure Lengend Snippet: Figure 1. m6A characteristics after MT treatment in aged mice (A) Representative ovarian histology of 2-month-old mice (young), MT-fed 12- month-old mice (aged + MT), and control 12-month-old mice (Aged). The primary and secondary follicle stages (marked with PF and SF, re spectively) are marked by black arrows. Scale bars, 200 μm (upper) and 20 μm (lower). (B) Quantification of different types of follicles in ovaries from young, aged, and aged + MT mice. Primordial, primary, secondary, corpus luteum, and antral follicles (marked with PmF, PF, SF, CL, and AF, respectively) were counted. n.s., not significant; *P < 0.05. (C) Quantification of the corpus luteum in ovaries from young, aged, and aged + MT mice. (D) Violin plot showing the level of m6A in the ovaries of young, aged and aged + MT mice. n = 5, **** P < 0.0001. (E) Volcano plot showing m6A-modified differentially expressed genes (DEGs) in the ovaries of aged and aged + MT mice (P value < 0.05 and fold change > 2). Down regulated (blue), upregulated (red), and unchanged (gray) genes are indicated. (F) Correlation analysis of 566 m6A DEGs among aged and aged + MT mice. (G) Gene Ontology (GO), KEGG, and Reactome (REAC) pathway analyses of m6A-modified differentially expressed genes. (H) GSEA pathway analysis of the cell cycle. (I,J) Immunofluorescence staining of ovarian GCs from young, aged and aged + MT mice, showing YTHDF2 (red) and DAPI (blue). Scale bar, 50 μm. ***P < 0.001.

Article Snippet: To generate YTHDF2 or UBE3C knockdown vectors, specific shRNAs were cloned and inserted into the pLKO.1 backbone (10878; Addgene, Watertown USA).

Techniques: Control, Modification, Immunofluorescence, Staining

Journal: Acta biochimica et biophysica Sinica

Article Title: Melatonin mitigates ovarian aging through regulation of the YTHDF2/m 6 A/UBE3C axis.

doi: 10.3724/abbs.2025090

Figure Lengend Snippet: Figure 2. MT downregulates m6A levels in KGN cells by increasing YTHDF2 expression (A,B) Cell senescence was detected by SA-β-galacto sidase (SA-β-gal) staining in KGN cells under different conditions: without H2O2 (NC), with H2O2 treatment (400 μM, 12 h), and with H2O2 treatment in the presence of MT (300 μM, 12 h). Scale bar, 40 μm. (C, D) KGN cell apoptosis was detected via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Scale bar, 50 μm. (E) m6A dot blot analysis to assess m6A levels in KGN cells under different conditions: without H2O2 (NC), with H2O2 treatment, and with H2O2 treatment in the presence of MT. (F,G) Western blot analysis showing YTHDF2 expression in KGN cells. (H, I) Western blot analysis of YTHDF2 expression in scramble control, YTHDF2 knockdown (shYTHDF2) and MT-treated (300 μM, 12 h) KGN cells in the absence of H2O2 exposure. (J, K) Cell senescence was detected by SA-β-gal staining in scramble control-, shYTHDF2- and MT-treated KGN cells in the absence of H2O2 exposure. Scale bar, 40 μm. (L,M) Cell senescence detected by SA-β-gal staining in NC, H2O2-treated, and YTHDF2- overexpressing KGN cells under H2O2 exposure. Scale bar, 40 μm. Dot blot analysis of m6A levels in scramble control, shYTHDF2- and MT-treated KGN cells in the absence of H2O2 exposure. *P < 0.05, ****P < 0.0001.

Article Snippet: To generate YTHDF2 or UBE3C knockdown vectors, specific shRNAs were cloned and inserted into the pLKO.1 backbone (10878; Addgene, Watertown USA).

Techniques: Expressing, Staining, TUNEL Assay, Dot Blot, Western Blot, Control, Knockdown

Journal: Acta biochimica et biophysica Sinica

Article Title: Melatonin mitigates ovarian aging through regulation of the YTHDF2/m 6 A/UBE3C axis.

doi: 10.3724/abbs.2025090

Figure Lengend Snippet: Figure 3. YTHDF2 regulates the m6A level of UBE3C RNA to affect the level of UBE3C expression (A) Correlation analysis of the m6A levels of ubiquitin protein ligases in aged and aged + MT mice. (B) MeRIP‒qPCR analysis showing the relative mRNA expression levels of ubiquitin-related genes in NC, H2O2 and H2O2+MT KGN cells. (C,D) Western blot analysis showing the expressions of YTHDF2, P53, and UBE3C in NC, H2O2 and H2O2 + MT KGN cells. (E,F) Western blot analysis showing the expression of YTHDF2 in scramble-, YTHDF2-knockdown- and MT-treated KGN cells under H2O2-untreated conditions. (G, H) Cell senescence was detected by SA-β-gal staining under H2O2-untreated conditions. Scale bar, 40 μm. (I,J) Cell senescence detected by SA-β-gal staining in NC, H2O2-treated, and UBE3C-overexpressing KGN cells under H2O2 exposure. Scale bar, 40 μm.

Article Snippet: To generate YTHDF2 or UBE3C knockdown vectors, specific shRNAs were cloned and inserted into the pLKO.1 backbone (10878; Addgene, Watertown USA).

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Knockdown, Staining

Journal: Acta biochimica et biophysica Sinica

Article Title: Melatonin mitigates ovarian aging through regulation of the YTHDF2/m 6 A/UBE3C axis.

doi: 10.3724/abbs.2025090

Figure Lengend Snippet: Figure 4. UBE3C upregulates P53 ubiquitination to delay aging (A) Western blot analysis was used to verify the level of P53 ubiquitination in the immunoprecipitates after H2O2 and MT treatment. (B,C) Western blot analysis was used to verify the expressions of P53 and UBE3C after UBE3C was knocked down. (D) qRT-PCR analysis showing the mRNA expression of P53 after UBE3C knockdown. n.s., not significant; ***P < 0.001; ****P < 0.0001. (E,F) Western blot analysis was used to detect the protein expressions of P53 and UBE3C in UBE3C-knockdown and YTHDF2-over expressing KGN cells. (G) Western blot analysis was used to verify the level of P53 ubiquitination in the immunoprecipitates of UBE3C-over expressing and YTHDF2-knockdown KGN cells treated with MG132. (H) Model of the mechanism by which melatonin alleviates ovarian aging in an m6A-dependent manner.

Article Snippet: To generate YTHDF2 or UBE3C knockdown vectors, specific shRNAs were cloned and inserted into the pLKO.1 backbone (10878; Addgene, Watertown USA).

Techniques: Ubiquitin Proteomics, Western Blot, Quantitative RT-PCR, Expressing, Knockdown

Journal: Biology direct

Article Title: MLL1 promotes placental trophoblast ferroptosis and aggravates preeclampsia symptoms through epigenetic regulation of RBM15/TRIM72/ADAM9 axis.

doi: 10.1186/s13062-024-00572-0

Figure Lengend Snippet: Fig. 3 MLL1 promotes RBM15 expression by increasing H3K4me3 modification; RBM15 enhances m6A modification to promote YTHDF2 binding to TRIM72 mRNA and degrade TRIM72, thereby inhibiting TRIM72 expression. A: Analysis of the enrichment of MLL1 and H3K4me3 on RBM15 promoter in tissues (n = 6) and cells (n = 3) by ChIP. B-C: Detection of RBM15 and TRIM72 expressions in tissues (n = 6) and cells (n = 3) by qRT-PCR and Western blot. D: Quantitative analysis of m6A modification in tissues (n = 6) and cells (n = 3). E: Analysis of the m6A modification on TRIM72 mRNA and the enrichment of YTHDF2 in tissues (n = 6) and cells (n = 3) by RIP. F: Detection of TRIM72 mRNA stability in cells (n = 3) by actinomycin D; HTR-8/SVneo cells were transfected with sh-YTHDF2, with sh-NC as the control. G-H: Detection of YTHDF2 expression in cells (n = 3) by qRT-PCR and Western blot. Data in panels AEF were analyzed by two-way ANOVA, and data in panels BCD were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. Data in panels GH were analyzed by t test. **P < 0.01

Article Snippet: R: T G A T A A A A G C C C A G C C C A G T A C Note: MLL1: mixed-lineage leukemia 1; RBM15: RNA binding motif protein 15; YTHDF2: YTH domain family 2; TRIM72: tripartite motif containing 72; ADAM9: ADAM metallopeptidase domain 9; GAPDH: glyceraldehyde-3-phosphate dehydrogenase nb600-256, NOVUS) and H3K4me3 (1:1000, ab213224, Abcam) at 4 °C overnight.

Techniques: Expressing, Modification, Binding Assay, Quantitative RT-PCR, Western Blot, Transfection, Control

Journal: Biology direct

Article Title: MLL1 promotes placental trophoblast ferroptosis and aggravates preeclampsia symptoms through epigenetic regulation of RBM15/TRIM72/ADAM9 axis.

doi: 10.1186/s13062-024-00572-0

Figure Lengend Snippet: Fig. 9 MLL1 promotes RBM15 expression by increasing H3K4me3 on the RBM15 promoter; RBM15 upregulates TRIM72 mRNA m6A modification, pro motes the binding between YTHDF2 and TRIM72, and then degrades TRIM72 mRNA and inhibits TRIM72 expression, thereby reducing the ubiquitination degradation of ADAM9, stabilizing the expression of ADAM9, and eventually promoting ferroptosis of trophoblasts and aggravating PE

Article Snippet: R: T G A T A A A A G C C C A G C C C A G T A C Note: MLL1: mixed-lineage leukemia 1; RBM15: RNA binding motif protein 15; YTHDF2: YTH domain family 2; TRIM72: tripartite motif containing 72; ADAM9: ADAM metallopeptidase domain 9; GAPDH: glyceraldehyde-3-phosphate dehydrogenase nb600-256, NOVUS) and H3K4me3 (1:1000, ab213224, Abcam) at 4 °C overnight.

Techniques: Expressing, Modification, Binding Assay, Ubiquitin Proteomics