Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: Mn treatment decreases YTHDF2 in cell culture (A) Primary mouse astrocytes were exposed to 100 μM for 1–24 h and YTHDF2 levels were determined by Western blot. The bottom panel shows the results of densitometric analysis of YTHDF2 bands normalized by β-actin (n = 4–5). YTHDF2 decreases time-dependently beginning after 3 h in primary mouse astrocytes. (B) ICC representation at 40x depicting YTHDF2 decreases in Mn-exposed primary mouse astrocytes at 24 h (100 μm scale). (C) 24-h treatment of human U251 astrocytes with different heavy metals, all at 100 μM ( n = 3). (D) Mn decreases YTHDF2 mRNA at 24 h in U251 astrocytes ( n = 5). (E) Mn decreases global m6A levels at 24 h in U251 astrocytes, as measured by LC-MS/MS ( n = 6). Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Cell Culture, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: YTHDF2 levels can affect pro-inflammatory chemokine/cytokine responses in Mn-exposed astrocytes (A) Validation of YTHDF2 knockdown using siRNA, both qPCR and immunoblotting (n = 5–6). (B) Validation of YTHDF2 overexpression, both qPCR and immunoblotting ( n = 3). (C) siYTHDF2 increased basal pro-inflammatory gene expression and exacerbated after Mn exposure ( n = 3). (D) Overexpression of YTHDF2 suppressed basal pro-inflammatory gene expression and prevented upregulation after Mn exposure ( n = 4). (E and F) Multiplex ELISA analysis of the treatment media of siYTHDF2 and YTHDF2 overexpression experiments for cytokines/chemokines (n = 4–6). IL-8 was most consistently affected by YTHDF2 levels, showing exacerbated release in siYTHDF2 experiments, while its release was prevented in YTHDF2 overexpression experiments. MCP1 (MCAF), IFNγ, IL-6, and MIP1b showed similar trends but with less consistency overall. Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence. Two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Biomarker Discovery, Knockdown, Western Blot, Over Expression, Gene Expression, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: RNA- and RIP-sequencing of Mn-exposed U251 astrocytes reveals YTHDF2 targeting of MAP2K4 (SEK1) (A and B) (A) Z-scores of Mn/Ctrl GO for Transcriptional Factor Targets, indicating important roles for cJUN and HIF1α (B) RIP-sequencing graphical representation of statistically significant YTHDF2 targets in Ctrl that were affected by Mn exposure. YTHDF2 targets are identified as having ≥+1 log2(RIP/input) ratio. MAP2K4 was identified as a lost YTHDF2 target under Mn exposure, suggesting regulation of the SEK1( MAP2K4 )-JNK-cJUN pathway. (C) si YTHDF2 astrocytes present with longer MAP2K4 mRNA half-life ( n = 3). (D) ICC representation at 40x depicting si YTHDF2 astrocytes have increased SEK1 protein levels (100 μm scale). (E) YTHDF2 overexpressing astrocytes present with shorter MAP2K4 half-life under Mn exposure (n = 4–5). (F) ICC representation at 40x depicting YTHDF2-overexpressing astrocytes have decreased SEK1 protein levels (100 μm scale). (G and H) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and sustained in siYTHDF2 Mn-exposed astrocytes. SEK1 protein levels are basally increased in siYTHDF2 astrocytes (n = 3–4). (I and J) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and prevented in YTHDF2-overexpressing Mn-exposed astrocytes. SEK1 protein levels are basally decreased in YTHDF2-overexpressing astrocytes ( n = 4). Data are means ± SEM. Two-group comparisons performed using unpaired t-test, with 2-fold gene threshold and 1.96 Z - score threshold using Altanalyze. Adjusted p -values < 0.05 considered significant evidence. For mRNA half-life comparisons, One-phase decay non-linear regression analysis was performed. p -values < 0.05 considered significant evidence. two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Sequencing, Western Blot, Phospho-proteomics, Comparison

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: YTHDF2 is decreased in Mn-gavaged mice, and a conditional knockout of YTHDF2 in the astrocytes of mice leads to increased astrocyte reactivity in substantia nigra pars compacta/reticulata and globus pallidus (A) IHC representation at 40x depicting YTHDF2 decreases and colocalization in GFAP+ cells in the globus pallidus of Mn-gavaged mice (100 μm scale). (B) Immunoblotting of the substantia nigra showing decreases of YTHDF2 in mice gavaged with Mn ( n = 9). (C) Schematic of Y2cKO mice generation. Tamoxifen (tiY2cKO) induces Cre recombination of YTHDF2 exon 4 to produce a band at 748 bp in astrocytes isolated from striatal/hippocampal brain tissue using biotinylated-EAAT1/GLAST-1 antibody. (D) Globus pallidus validation of YTHDF2 protein reduction and increased GFAP immunoreactivity, and also increased m6A staining upon YTHDF2 deletion. YTHDF2 was quantified by parent-child extraction using GFAP as the parent (n = 3–4) (100 μm scale). (E) Globus pallidus representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). (F) Immunoblotting and quantification of substantia nigra- showing tiY2cKO mice with increased GFAP immunoreactivity similar to Mn-gavaged mice, indicating specific loss of YTHDF2 in astrocytes increases their reactivity (n = 4–6). (G) Substantia nigra representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). Data are means ± SEM. Student’s t test or two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence, with q-values between 0.05 and 0.01 considered as weaker evidence taking into consideration variations in data and trends.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Knock-Out, Western Blot, Isolation, Biomarker Discovery, Staining, Extraction, Comparison

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: Integrated working hypothesis of YTHDF2’s role on the SEK1( MAP2K4 )-JNK-cJUN pathway Upon Mn exposure, ALKBH5 levels increase while YTHDF2 decreases, leading to increased half-life of MAP2K4 mRNA. Increased abundance of MAP2K4 leads to more SEK1 protein, allowing for the sustained activation (increased phosphorylation) of the downstream pathway targets JNK and cJUN by Mn, which promotes the pro-inflammatory response in astrocytes. When YTHDF2 is overexpressed, MAP2K4 mRNA is degraded. This leads to lower SEK1 protein levels, reducing the pathway activation of JNK and cJUN (decreased phosphorylation) by Mn, resulting in an anti-inflammatory response.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Activation Assay, Phospho-proteomics

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet:

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Recombinant, Extraction, Selection, Negative Control, Software

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