Journal: Advanced Science

Article Title: Multi‐Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

doi: 10.1002/advs.202301538

Figure Lengend Snippet: YTHDF3 deficiency inhibits RNA TE in mouse oocytes. a) The in vitro PB1 emission rates of mouse oocytes from the control groups and the m6A‐related gene depletion groups. Each dot represents a single biological replicate. p ‐Values were calculated with Student's t‐test for paired samples. b) Immunofluorescence verifying the depletion of YTHDF3 by Trim‐Away. Scale bar, 50 µm. The right panel shows the quantification of YTHDF3 protein levels. The average intensity of the control group oocytes was set as 1.0. Each dot represents a single oocyte analyzed. p ‐Value was calculated with two‐tailed Mann–Whitney test. c) Immunofluorescence verifying the expression of YTHDF3 in young and aged mouse GV oocytes. Scale bar, 50 µm. The right panel shows the quantification of YTHDF3 protein levels. The average intensity of young mouse oocytes was set as 1.0. Each dot represents a single oocyte analyzed. p ‐Value was calculated with two‐tailed Mann–Whitney test. d) Scatter plot showing the changes in gene translation and transcription in YTHDF3‐KD oocytes and control oocytes. The Pearson correlation coefficient = −0.196. e) Cumulative distribution of total RNA expression (log 2 TPM); the red line denotes the control group, and the blue line represents the YTHDF3‐KD group. f) Cumulative distribution of TE. The red line denotes the control group, and the blue line represents the YTHDF3‐KD group. g) Scatter plot showing the RNA TE alterations of YTHDF3‐KD oocytes compared with the control group. Red and blue dots denote up‐ and down‐regulated genes, respectively. Upregulated, FC>1.5; downregulated, FC<0.67. h) Gene set enrichment analysis of TE showing the TE downregulated genes enriched in the hallmark of the G2/M checkpoint and TE upregulated genes enriched in the hallmark of oxidative phosphorylation. i) Bar plots showing the numbers of high‐TE genes (TE>2) and low‐TE genes (TE<0.5) in the YTHDF3‐KD group and the control group oocytes, respectively. PB1, polar body‐1. TE, translational efficiency. FC, fold change. YTHDF3‐KD, YTHDF3 knockdown. Ns, no significant difference. * p < 0.05, **** p < 0.0001.

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955‐1‐AP),

Techniques: In Vitro, Control, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Expressing, RNA Expression, Phospho-proteomics, Knockdown

Journal: Advanced Science

Article Title: Multi‐Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

doi: 10.1002/advs.202301538

Figure Lengend Snippet: YTHDF3 modulates RNA translation efficiency in an m6A‐dependent manner. a) Gene set enrichment analysis demonstrating that the TE of m6A‐enriched RNA was significantly decreased upon YTHDF3 depletion. b) Bar plots showing the numbers of up‐ (FC>1.5) and down‐regulated (FC<0.67) genes for m6A‐enriched genes or genes not enriched by m6A, respectively. Pink denotes m6A‐enriched genes. Blue denotes genes not enriched by m6A. c) Venn diagram portraying the overlap of YTHDF3 target genes among three independent RIP‐seq biological replicates. d) Motif identified by HOMER within YTHDF3 RIP‐seq peaks in HEK293T cells. e) Gene set enrichment analysis showing the TE alterations of YTHDF3‐binding RNA upon YTHDF3 depletion. f) Gene set enrichment analysis showing the TE alterations of m6A‐enriched YTHDF3‐binding RNA upon YTHDF3 depletion. g) Venn diagram showing the overlap of m6A‐modified YTHDF3 target genes between the differential TE genes in YTHDF3‐KD oocytes and the differential TE genes in aged mouse oocytes. h) The RNA TE log 2 fold change in the 449 overlapping genes (described in g) in aged mouse oocytes and YTHDF3‐depleted oocytes. i) The RNA translational level changes in 302 downregulated TE genes (described in h) in aged mouse oocytes and YTHDF3‐depleted oocytes. j) Immunofluorescence verifying the expression of HELLS in the control group and the YTHDF3‐depleted group oocytes. Scale bar, 50 µm. A screenshot of the nucleus is shown separately at the bottom. The right panel shows the quantification of the HELLS protein level. The average intensity of the control group oocytes was set as 1.0. Each dot represents a single oocyte analyzed. p ‐Value was calculated with two‐tailed Mann‐Whitney test. k) Schematic representation of wild‐type (YTHDF3‐WT) and mutant (YTHDF3‐Mut) YTHDF3 constructs. l) Western blot demonstrating the expression of HELLS in HEK293T cells transfected with empty vector or wild‐type or mutant Flag‐tagged YTHDF3 plasmid. GAPDH was used as the negative control. The left panel presents a representative Western blot image. The right panel shows the quantification of the HELLS protein level. The average intensity of the NC group was set as 1.0. Each dot represents a single biological replicate. p ‐Value was calculated with two‐tailed Mann–Whitney test. m) HEK293T cells were cotransfected with NC, YTHDF3‐WT, or YTHDF3‐Mut plasmids, and luciferase reporter plasmids carrying the HELLS 3’UTR, and luciferase activity was measured. p‐ Value was calculated with two‐tailed Mann–Whitney test. TE, translational efficiency. FC, fold change. HOMER, Hypergeometric Optimization of Motif EnRichment. RIP, RNA immunoprecipitation. YTHDF3‐KD, YTHDF3 knockdown. NC, negative control. Ns, no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955‐1‐AP),

Techniques: Binding Assay, Modification, Immunofluorescence, Expressing, Control, Two Tailed Test, MANN-WHITNEY, Mutagenesis, Construct, Western Blot, Transfection, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, RNA Immunoprecipitation, Knockdown

Journal: Advanced Science

Article Title: Multi‐Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

doi: 10.1002/advs.202301538

Figure Lengend Snippet: The changes of RNA translation efficiency in aged human oocytes. a) Scatter plot showing the changes in gene translation and transcription during oocyte aging. b) Bar plots showing the numbers of upregulated TE genes and downregulated TE genes in young and aged mouse/human GV oocytes, respectively. Upregulated, FC>1.5; downregulated, FC<0.67. c) Bar plots showing the numbers of high‐TE genes (TE>2) and low‐TE genes (TE<0.5) in young and aged mouse/human GV oocytes, respectively. d) Violin plots showing the TE changes in the genes of the group not enriched by m6A and of the m6A‐enriched group in aged human oocytes compared with young human oocytes. p ‐Value was calculated with Student's t‐test for independent samples. e) Gene set enrichment analysis demonstrating the correlation of TE alterations and YTHDF3 target RNA in aged human oocytes. f) Violin plots showing the TE changes in genes of the papCPE‐containing group and in genes of the group not containing papCPE in aged human oocytes compared with young human oocytes. p ‐Value was calculated with Student's t‐test for independent samples. g) Violin plots showing the TE changes in genes of the CPE‐containing group and in genes of the group not containing CPE in aged human oocytes compared with young human oocytes. p ‐Value was calculated with Student's t‐test for independent samples. h) Violin plots showing the TE changes in four groups of genes in aged human oocytes compared with young human oocytes. p ‐Values were calculated with one‐way ANOVA and Bonferroni post hoc test. i) Violin plots showing the TE changes in four groups of genes in aged human oocytes compared with young human oocytes. p ‐Values were calculated with one‐way ANOVA and Bonferroni post hoc test. j) Transcriptional and translational expression levels of the YTHDF3 in human/mouse GV oocytes. Data are shown as the mean ± SEMs. p ‐Values were calculated with Student's t‐test for independent samples. k) Transcriptional and translational expression levels of the HELLS in human/mouse GV oocytes. Data are shown as the mean ± SEMs. p‐Values were calculated with Student's t‐test for independent samples. l) Representative RBPs enriched in 3’UTR of the genes in human oocytes that potentially regulated RNA TE. Data are shown as the means±SEMs. p ‐Values were calculated with Student's t‐test for independent samples. FC, fold change. TE, translational efficiency. CPEs, cytoplasmic polyadenylation elements. papCPE, CPEs within 100 bp of PASs. RBPs, RNA binding proteins. Ns, no significant difference. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955‐1‐AP),

Techniques: Expressing, RNA Binding Assay

Journal: Discover. Oncology

Article Title: Celastrol suppresses human pancreatic cancer via m 6 A-YTHDF3-mediated downregulation of Claspin and Bcl-2.

doi: 10.1007/s12672-023-00838-5

Figure Lengend Snippet: Fig. 3 Celastrol treatment regulated the expression of Claspin, Bcl-2, METTL3, and YTHDF3 in pancreatic cancer cells. A, B Clustered heat- map of the differentially expressed genes in AsPC-1 cells in the presence or absence of celastrol (both in triplicate) by RNA-seq analysis (A), and the volcano plots of the differentially expressed genes were shown (B). C, D RT-qPCR (C) and Western blotting (D) were applied to validate the expression of Claspin, Bcl-2, METTL3, and YTHDF3 in AsPC-1 and BxPC-3 cells in the presence or absence of celastrol (Cel). GAPDH was used as a loading control. E RT-qPCR was performed to validate the expression of Claspin, Bcl-2, METTL3, and YTHDF3 in xeno- graft tumor tissues in indicated celastrol (Cel) administration groups. F, G Representative immunohistochemical staining and immunohisto- chemical score for Claspin, Bcl-2, METTL3, and YTHDF3 in tissue sections of tumors from indicated celastrol (Cel) administration groups was shown. Data were shown as the means ± SD from three independent experiments, *P < 0.05. Scale bars = 20 µm

Article Snippet: The antibodies against METTL3 (#86132), Claspin (#2800), YTHDF3 (#24206), GAPDH (#51332), m6A (#56593), and HRP-coupled anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Discover. Oncology

Article Title: Celastrol suppresses human pancreatic cancer via m 6 A-YTHDF3-mediated downregulation of Claspin and Bcl-2.

doi: 10.1007/s12672-023-00838-5

Figure Lengend Snippet: Fig. 6 Celastrol down-regulated Claspin and Bcl-2 expression in an m6A-YTHDF3 mediated pattern in pancreatic cancer cells. A, B The rela- tive levels of Bcl-2 or Claspin mRNA in celastrol-treated both AsPC-1 (A) and BxPC-3 cells (B) after incubation with actinomycin D (2 μg/ ml) for the indicated times (normalized to 0 h) were determined by RT-qPCR. C RIP-qPCR assay was performed to evaluate the interaction between YTHDF3 and the mRNA of Claspin or Bcl-2 in AsPC-1 and BxPC-3 cells in the presence or absence of celastrol. D, E The relative lev- els of Claspin or Bcl-2 protein in celastrol-treated AsPC-1 and BxPC-3 cells after incubation with cycloheximide for the indicated times were determined by Western blotting (D) and quantitatively analyzed (E). All data were shown as the means ± SD from three independent experi- ments, *P < 0.05

Article Snippet: The antibodies against METTL3 (#86132), Claspin (#2800), YTHDF3 (#24206), GAPDH (#51332), m6A (#56593), and HRP-coupled anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot

Journal: MedComm

Article Title: N6‐methyladenosine reader YTHDF3‐mediated zinc finger protein 41 inhibits hepatocellular carcinoma progression by transcriptional repression of Snail

doi: 10.1002/mco2.763

Figure Lengend Snippet: YTHDF3‐mediated m 6 A modification of ZFP41 mRNA and decays its mRNA stability. (A) qPCR results showed that efficiencies of siRNA‐mediated knockdown of common m 6 A regulators in MHCC‐97H cells. (B) qPCR results showed that expression of ZFP41 after silencing these m 6 A regulators in MHCC‐97H cells. (C) Overexpression of YTHDF3 notably suppress ZFP41 expression on transcription level in MHCC‐97H and Hep3B cells. (D) Knockdown of YTHDF3 obviously increase ZFP41 expression on mRNA level in MHCC‐97H and Hep3B cells. (E) Western blots results demonstrated that the ZFP41 protein level in MHCC‐97H and HLF cells after silencing YTHDF3 with siRNA of YTHDF3. (F) Western blots results demonstrated that the ZFP41 protein level in Hep3B cells after overexpression of YTHDF3. (G) The diagram of the potential site of m 6 A modification on the CDS (Coding Sequence) area. (H) The luciferase activity in both MHCC‐97H and Hep3B cells cotransfected with relative plasmids. (I) MeRIP results showed that ZFP41‐wt or ZFP41‐mut in both MHCC‐97H and Hep3B cells. (J) The rate of ZFP41 mRNA degradation in MHCC‐97H and Hep3B cells with YTHDF3 overexpression or knockdown. (K) The binding of ZFP41 mRNA and YTHDF3 was tested in MHCC‐97H and Hep3B cells by RIP‐qPCR analyses. (L) The relationship between ZFP41 and YTHDF3 was confirmed by RNA pull‐down assay.

Article Snippet: Antibodies for YTHDF3 (25537‐1‐AP), E‐cadherin (20874‐1‐AP), N‐cadherin (22018‐1‐AP), and GAPDH (60004‐1‐Ig) were supplied by Proteintech.

Techniques: Modification, Knockdown, Expressing, Over Expression, Western Blot, Sequencing, Luciferase, Activity Assay, Binding Assay, Pull Down Assay

Journal: MedComm

Article Title: N6‐methyladenosine reader YTHDF3‐mediated zinc finger protein 41 inhibits hepatocellular carcinoma progression by transcriptional repression of Snail

doi: 10.1002/mco2.763

Figure Lengend Snippet: Schematic diagram of the mechanism showing the roles played by ZFP41 in HCC cells. In normal liver cells, YTHDF3 shows low expression, leading to a reduction in the m6A modification of ZFP41. This results in increased synthesis of ZFP41 mRNA and an enhanced transcriptional inhibitory effect on Snail. In contrast, in HCC cells, the expression of YTHDF3 is significantly elevated, which enhances the m6A modification of ZFP41. This promotes degradation of ZFP41 mRNA, consequently weakening the transcriptional inhibitory effect on Snail. Ultimately, this facilitates activation of the EMT pathway and promotes proliferation and metastasis of HCC.

Article Snippet: Antibodies for YTHDF3 (25537‐1‐AP), E‐cadherin (20874‐1‐AP), N‐cadherin (22018‐1‐AP), and GAPDH (60004‐1‐Ig) were supplied by Proteintech.

Techniques: Expressing, Modification, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms

doi: 10.3389/fphar.2023.1260300

Figure Lengend Snippet: FTO regulates FOXO6 m6A modification via YTHDF3-dependent manner. (A) Effect of FTO overexpression and knockdown on m6A level of FOXO6 detected by MeRIP-qPCR; (B) The potential m6A sites in FOXO6 predicted by SRAMP website; (C) The secondary RNA structure and location of m6A site on FOXO6 mRNA; (D) The m6A level of FOXO6 detected by MeRIP-qPCR after methylation site mutation; (E) Effect of FTO overexpression on m6A level of FOXO6 detected by MeRIP-qPCR after methylation site mutation; (F) The expression level of m6A recognition protein YTHDF3 detected by Western blot (1:Control; 2:Model); (G) RIP‐qPCR detection of the binding relationship between YTHDF3 and FOXO6; (H) The effect of YTHDF3 knockdown on FOXO6 expression detected by RT-qPCR; (I) The effect of YTHDF3 knockdown on FOXO6 mRNA stability detected by actinomycin D assay. * p < 0.05 ; ** p < 0.01 .

Article Snippet: HGMC lysate samples were incubated with either YTHDF3 antibody (IP) (0202730101, ABclonal, Wuhan, China) or control IgG antibody (IgG) at 4°C for 16 h. Finally, total RNA was extracted and analysed using RT-qPCR assay.

Techniques: Modification, Over Expression, Knockdown, Methylation, Mutagenesis, Expressing, Western Blot, Control, Binding Assay, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms

doi: 10.3389/fphar.2023.1260300

Figure Lengend Snippet: The binding relationship between YTHDF3 and FOXO6 predicted by RM2Target website.

Article Snippet: HGMC lysate samples were incubated with either YTHDF3 antibody (IP) (0202730101, ABclonal, Wuhan, China) or control IgG antibody (IgG) at 4°C for 16 h. Finally, total RNA was extracted and analysed using RT-qPCR assay.

Techniques: Binding Assay, Modification