Journal: Cancer letters

Article Title: m 6 A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition

doi: 10.1016/j.canlet.2023.216178

Figure Lengend Snippet: YTHDF3 specifically interacts with DRACH RNA motifs in cyclin D1 and c-myc IRESs to regulate IRES activity and mTOR inhibitor sensitivity. (A) Screening of YTH domain family proteins binding to the c-myc DRACH RNA motifs (motifs 1–3, see fig. 2A) in U373MG(Uppsala) or HK296 GBM cells in the absence or presence of PP242 as indicated (100 nM, 18 h). The indicated proteins were immunoprecipitated from lysates and bound RNAs were amplified using primers specific for the hairpin sequences. + S.D., n = 4. (B) as in (A) except binding to the two CCND1 DRACH RNA motifs (motifs 1–2, see fig. 2A) were assessed. (C) Induction of YTHDF3 protein expression following PP242 exposure (100 nM, 18 h) in the indicated lines. (D) siRNA mediated knockdown of YTHDF3 sensitizes PP242 resistant GBM lines to PP242 (100 nM, 48 h). ATP-release assays were performed (Promega CellTiter-Glo® assay) and cell proliferation determined as a percentage of control vehicle treated cells +S.D.; n = 3. (E) siRNA mediated knockdown of YTHDF3 inhibits PP242 induced IRES activity. U373MG(Uppsala) or HK296 cells were transfected with the indicated non-targeting control (scrambled sequence; shscr) or two individual (siYTHDF3–1 or −2) YTHDF3 targeting siRNAs and IRES activity determined from CCND1 or c-MYC dicistronic mRNA reporters in the absence or presence of PP242 as indicated. Luciferase activity was normalized to the luciferase mRNA level. * P-values are shown. Mean + S.D., n = 3.

Article Snippet: Site-specific m 6 A mutagenesis was performed using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) with the appropriate mutagenic primers to generate c-myc and cyclin D1 IRES luciferase reporter constructs. shRNA constructs (psi-LVRH1P) targeting human YTHDF3 and a scrambled sequence control were obtained from GeneCopoeia (#HSH069323).

Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Amplification, Expressing, Knockdown, Glo Assay, Control, Transfection, Sequencing, Luciferase

Journal: Cancer letters

Article Title: m 6 A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition

doi: 10.1016/j.canlet.2023.216178

Figure Lengend Snippet: hnRNP A1 and YTHDF3 interact and hnRNP A1-induced nucleic acid annealing activity is stimulated by YTHDF3. (A) Extracts from U373MG(Uppsala) cells were immunoprecipitated with nonspecific IgG (control) or antibody against YTHDF3 (left panel) or hnRNP A1 (right panel) and immunoprecipitates immunoblotted for the indicated proteins. Input lysates were probed for the indicated proteins as shown. (B) Recombinant YTHDF3 or hnRNP A1 was purified and analyzed for reannealing activity. 1 pmol of each protein was used in the annealing reactions and the migration positions of the indicated species are displayed.

Article Snippet: Site-specific m 6 A mutagenesis was performed using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) with the appropriate mutagenic primers to generate c-myc and cyclin D1 IRES luciferase reporter constructs. shRNA constructs (psi-LVRH1P) targeting human YTHDF3 and a scrambled sequence control were obtained from GeneCopoeia (#HSH069323).

Techniques: Activity Assay, Immunoprecipitation, Control, Recombinant, Purification, Migration

Journal: Frontiers in Neurology

Article Title: Identification of m1A/m6A/m5C/m7G-related genes and clusters associated with neuropathic pain

doi: 10.3389/fneur.2026.1592545

Figure Lengend Snippet: Validation of expression of the six m1A/m6A/m5C/m7G-related DEGs ( FTO , METTL3 , NSUN2 , YTHDF3 , WDR4 , and EIF4E ) in rats after 3, 7, and 14 days of SNL surgery. n = 3 per group. SNL, spinal nerve ligation. (A) qPCR results. (B) Western blot analysis results. * p < 0.05.

Article Snippet: After blocking, the membranes were incubated with the following primary antibodies: Nsun2 (1:1000, PH6626, ab-mart, Shanghai, China), Mettl3 (1:1000, 15,073-1-AP, Proteintech, Rosemont, IL, United States), Ythdf3 (1:500, 25,537-1-AP, Proteintech), FTO (1:1000, PA2776, ab-mart); Wdr4 (1:1000, PS17092, ab-mart), Eif4e (1:1000, 11,149-1-AP, Proteintech), and GAPDH (1:20000, 10,494-1-AP, Proteintech) overnight at 4 °C.

Techniques: Biomarker Discovery, Expressing, Ligation, Western Blot

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: Cell reports

Article Title: Ythdf m 6 A Readers Function Redundantly during Zebrafish Development

doi: 10.1016/j.celrep.2020.108598

Figure Lengend Snippet: (A) Numbers of male and female fish of each genotype. Sibling control and double ythdf2;ythdf3 homozygotes were offspring from the same cross, depicted on top. (B) Gonad histology of double homozygous ( ythdf2 −/− ; ythdf3 −/− ) and sibling control fish from the cross in (A). At 27 dpf, mutants exhibit less developed juvenile ovaries than controls. At 35 dpf, 6 sibling fish had adult ovaries and 8 had testes, while all 12 ythdf2 −/− ; ythdf3 −/− fish had testes. I, stage I oocytes; II, stage II oocytes; triangle, apoptotic oocyte; sg, spermatogonia; sc, spermatocytes. n, replicate number with similar gonads. Scale bars, 40 μm. (C) Numbers of male and female fish of each genotype, following treatment with 17α-ethynylestradiol (EE2). Fish were from the same cross as in (A).

Article Snippet: For gene editing to generate ythdf2 −223/−223 and ythdf3 mutants, 30 pg of each sgRNA was co-injected with 150 pg of Cas9 (plasmid pT3TS-nCas9n, Addgene #46757, ( )) capped mRNA synthesized using mMessage mMachine T3 Transcription kit (Thermo Fisher Scientific, AM1340).

Techniques: Control

Journal: Cell reports

Article Title: Ythdf m 6 A Readers Function Redundantly during Zebrafish Development

doi: 10.1016/j.celrep.2020.108598

Figure Lengend Snippet: (A) MZ ythdf2 ;MZ ythdf3 , background-matched wild-type, and unrelated TU-AB wild-type zebrafish embryos develop at similar rates. Parents of mutant and background-matched control embryos were 17α-ethynylestradiol treated. n, replicate number of embryos at same developmental stage. Scale bars, 500 μm. (B) Biplot of expression (log 2 RPKM) of maternal (n = 13,642) and m 6 A-modified (n = 2,280) mRNAs between wild-type and MZ ythdf2 ;MZ ythdf3 , from 6 hpf poly(A) mRNA-seq. Dashed lines, 2-fold change. (C) Cumulative distribution of fold changes in maternal mRNA abundance (log 2 RPKM) between 4 and 0 hpf in MZ ythdf2 ;MZ ythdf3 embryos, for m 6 A-modified (n = 708) and non-modified (n = 841) mRNAs, from poly(A) mRNA-seq. p values computed by a Mann-Whitney U test. (D) Schematic of cross and genotyping strategy for triple Ythdf mutants. Female fish ( ythdf1 +/− ; ythdf2 −/− ; ythdf3 +/− ) were crossed to males ( ythdf1 −/− ; ythdf -+/− ; ythdf3 −/− ) to generate triple homozygotes (1 of 8 possible genotypes). Every 3 days, 48 larvae were genotyped, with 200 more fish genotyped at 30 dpf. (E) Percentage of triple heterozygous (het) or triple homozygous (homo) fish during development. Dotted line, expected percentage (12.5%) of each genotype, from cross in (D). (F) Number of fish with each genotype from cross in (D) at 30 dpf. For each ythdf allele: filled circle, heterozygous; m, homozygous. Dotted line, expected fish number (25), equal for all genotypes.

Article Snippet: For gene editing to generate ythdf2 −223/−223 and ythdf3 mutants, 30 pg of each sgRNA was co-injected with 150 pg of Cas9 (plasmid pT3TS-nCas9n, Addgene #46757, ( )) capped mRNA synthesized using mMessage mMachine T3 Transcription kit (Thermo Fisher Scientific, AM1340).

Techniques: Mutagenesis, Control, Expressing, Modification, MANN-WHITNEY

Journal: Cell reports

Article Title: Ythdf m 6 A Readers Function Redundantly during Zebrafish Development

doi: 10.1016/j.celrep.2020.108598

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For gene editing to generate ythdf2 −223/−223 and ythdf3 mutants, 30 pg of each sgRNA was co-injected with 150 pg of Cas9 (plasmid pT3TS-nCas9n, Addgene #46757, ( )) capped mRNA synthesized using mMessage mMachine T3 Transcription kit (Thermo Fisher Scientific, AM1340).

Techniques: Recombinant, Nick Translation, SYBR Green Assay, RNA Extraction, Reverse Transcription, Mutagenesis, Software

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: a , Strategy for generating Ythdf3 knockout (KO) allele. F1 and R1: genotyping primers b , Quantitative PCR analysis of Ythdf3 mRNA levels in tissues from male Ythdf3 +/+ and Ythdf3 −/− mice (n = 3). c , Representative immunoblots showing expression of YTHDF3 in different tissues from male Ythdf3 +/+ and Ythdf3 −/− mice.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: Representative images of H&E-stained heart sections of male Ythdf3 +/+ and Ythdf3 −/− mice at 3 months (m) of age. Scale bar, 1 mm. b, Representative images of wheat germ agglutinin (WGA)-stained left ventricular muscle of male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m. Scale bar, 200 μm. Right: quantification of cell size, as determined by WGA staining. 500 cells from 5 mice per group were counted. c, Echocardiographic analysis showing left ventricle ejection fraction (EF), fractional shortening (FS) and left ventricular (LV) volume (s: systolic; d: diastolic) of male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m (n = 12). d, Left, representative images illustrating gross morphology of brains. Right, quantification of brain weight (bW)/body weight (BW) of male mice aged 3 m (n = 6). e, Representative images of Nissl-stained brain sections from male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m (top). Scale bar, 200 μm. Representative mages of periodic acid–Schiff (PAS)-stained brain sections from male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m (bottom). Scale bar, 200 μm. f, Heatmap of activity of mice in the Morris water maze 24 h post-training (n = 12 per genotype). g, Time mice spent in the quadrant and platform in the Morris water maze 24 h post-training (n = 12 per genotype). h, Representative images of H&E-stained skeletal muscle sections from male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m (top). Scale bar, 200 μm. Quantification of cross-section area of H&E staining (100 cells from 6 mice per group were counted) (bottom). i, Representative images of acetylcholinesterase (AChE) staining in skeletal muscle sections in male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m. Scale bar, 200 μm. j, Running endurance of male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m, as tested via treadmill (n = 10). Data represent the means ± SD. P -values were calculated by two-tailed unpaired Student’s t -test.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Staining, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: a , Representative images of H&E-stained heart sections of 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Scale bar, 1 mm. b , Top: representative images of WGA-stained left ventricular muscle sections of 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Bottom: representative images of Masson’s trichrome-stained heart sections from 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Scale bar, 200 μm. Right: quantification of cardiac cell size in WGA-stained sections; 500 cells from 5 mice per group were counted. c , Echocardiography data of 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice (n = 10-11). d , Left, representative images of murine brains. Right, brain weight (bW) and body weight (BW) of female mice aged 3 m (n = 6). e , Heatmap of activity of female mice in the Morris water maze 24 h after training (n = 6). f , Time female mice spent in the quadrant and platform in the Morris water maze 24 h after training (n = 6). g , Heatmap of activity of female mice in the Morris water maze 72 h after training (n = 6). h, Time female mice spent in the quadrant and platform in the Morris water maze 72 h after training (n = 6). i, Top: representative images of Nissl-stained brain sections of 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Bottom: representative mages of periodic acid–Schiff (PAS)-stained brain sections from 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Scale bar, 200 μm. j , Top: representative images of H&E-stained skeletal muscle sections from 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Scale bar, 200 μm. Bottom: quantification of cross-section area of H&E staining (100 cells from 6 mice per group were counted). k , Top: representative images of acetylcholinesterase (AChE) staining in skeletal muscle sections from 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Bottom: representative images of Masson’s trichrome-stained skeletal muscle sections from 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice. Scale bar, 200 μm. l , Running endurance of 3-m-old female Ythdf3 +/+ and Ythdf3 −/− mice, as tested via treadmill (n = 6). Data represent the means ± SD. P -values were calculated by two-tailed unpaired Student’s t -test.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Staining, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: a , Representative images of Masson’s trichrome-staining of heart, liver, muscle, kidney sections of 3-m-old male Ythdf3 +/+ and Ythdf3 −/− mice (left). Quantification of collagen-volume fraction (CVF%) in left (right). b , Representative images of immunostaining of α-SMA in heart, liver, kidney, and lung sections of 3-m-old male Ythdf3 +/+ and Ythdf3 −/− mice. c , Representative images of immunohistochemistry of Collagen I and Ⅲ in heart sections of 3-m-old male Ythdf3 +/+ and Ythdf3 −/− mice. d , Quantitative PCR analysis of collagen-related fibrosis genes in cardiac fibroblasts (P4) treated with vector, FLAG-YTHDF3 and -YTHDF3Mut (W438A and W492A mutations) lentivirus particles (n = 3 biological replicates). e-f , mRNA levels of the 67 genes that bound to YTHDFs ( Table S1 and ), determined by qRT-PCR in the anti-FLAG RIP immunoprecipitates in HEK293T cells. ( e ) Heatmap depicting relative levels of mRNAs bound to YTHDFs. ( f ) RIP-PCR showing top 10 mRNAs with the strongest binding capacity to YTHDF3 (n = 3 biological replicates). g , RIP-PCR analysis of mRNAs from f in HEK293T cells overexpressing FLAG-YTHDF3 and -YTHDF3M (W438A and W492A). GJA1 was included as positive control , and GAPDH was included as negative control. n = 3 biological replicates. Data represent the means ± SD. P -values were calculated by two-tailed unpaired Student’s t -test. Scale bar, 200 μm.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Staining, Immunostaining, Immunohistochemistry, Real-time Polymerase Chain Reaction, Plasmid Preparation, Quantitative RT-PCR, Binding Assay, Positive Control, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: Representative immunoblots showing proteins in anti-HA immunoprecipitates and whole-cell lysate (WCL) of HEK293T cells transfected with FLAG-YTHDF3 and/or LAMP2B-HA. b, Representative images of immunohistochemical staining (IHC) of p-S6 in tissues from 3-m-old male Ythdf3 +/+ and Ythdf3 −/− mice. Scale bar, 200 μm. c, Representative immunoblots showing protein expression in tissues from male Ythdf3 +/+ and Ythdf3 −/− mice aged 25 m.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Western Blot, Transfection, Immunohistochemical staining, Staining, Paraffin-embedded Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: a , Venn diagram of the overlap among transcriptomes associated with fibrotic diseases (blue circle) , m6A-modified RNAs in mouse heart tissues (green circle, GSE201764), and YTHDF3-bound transcripts in murine primary hippocampal neurons (purple circle, GSE194207). b , Anti-FLAG RIP followed by quantitative PCR analysis of mRNAs in HEK293T cells overexpressing FLAG-YTHDFs (n = 3 biological replicates).

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Modification, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: a , Quantitative PCR analysis of Sox9 mRNA levels in heart tissues of male Ythdf3 +/+ and Ythdf3 −/− mice aged 3 m. b , Representative fluorescence images showing Sox9 protein in heart tissues of 3-m-old male Ythdf3 +/+ and Ythdf3 −/− mice (n = 4). Scale bar, 30 μm. c , Quantitative PCR analysis of Sox9 downstream gene levels in heart tissues of 3-m-old male Ythdf3 +/+ and Ythdf3 −/− mice (n = 4). d , Sox9 mRNA degradation analysis in Ythdf3 +/+ and Ythdf3 −/− cardiac fibroblast. Insert: comparation of calculated half-life of Sox9 mRNAs. e , YTHDF3-RIP followed by quantitative PCR analysis of SOX9 and SOX9 mutation (m6A sites) mRNA levels in HEK293T cells overexpressing SOX9 and its mutation (n = 3 biological replicates). f , Degradation analysis of endogenous Sox9 , ectopic truncated Sox9 (t Sox9 ) and its m6A mutation (t Sox9m ) in male Ythdf3 +/+ and Ythdf3 −/− cardiac fibroblasts overexpressing the t Sox9 or t Sox9m (n = 3 biological replicates). Left: detection of endogenous Sox9 ; right: detection of t Sox9 and t Sox9m . Insert: half-life analysis of Sox9 variants. g , Representative fluorescence images showing Sox9 -, Sox9 with m6A mutation ( Sox9m ) - 12×MS2/MCP-EGFP and Ytthdf3 (red) in cardiac fibroblasts. Scale bar, 15 μm. h , Representative fluorescence images showing Sox9 mRNA (Cy3) and Ythdf3 (green) in mouse cardiac fibroblasts transfected with sh Scr or sh Mettl3 . Scale bar, 15 μm. i , Quantitative PCR analysis of Sox9 mRNA levels in scramble (shScr) and sh Mettl3 shRNA-transfected cardiac fibroblasts (n = 3 biological replicates). j , Representative immunoblots showing SOX9 protein level in cardiac fibroblasts from male Ythdf3 +/+ and Ythdf3 −/− mice after treatment with cycloheximide (CHX) (50 µg/mL); chloroquine (CQ, 50 μM); or MG132 (10 μM). Right: Quantification of the SOX9 protein level was shown. Data represent the means ± SD. P -values were calculated by two-tailed unpaired Student’s t -test.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Real-time Polymerase Chain Reaction, Fluorescence, Mutagenesis, Transfection, shRNA, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: Quantitative PCR analysis of Sox9 downstream gene levels in Ythdf3 +/+ and Ythdf3 −/− cardiac fibroblast cells (P4) treated with vector, FLAG-YTHDF3 and -YTHDF3Mut (W438A and W492A) lentivirus particles (n = 3 biological replicates). Data represent the means ± SD. P -values were calculated by two-tailed unpaired Student’s t -test.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Ythdf3 causes Danon disease-like features

doi: 10.1101/2025.06.10.658803

Figure Lengend Snippet: Design of AAV-sh Sox9 therapeutic study. Injection was administered at 4 months and testing was conducted at 5 months. b, Quantitative PCR analysis of Sox9 mRNA levels in heart and brain tissues after AAV- shSox9 therapy. c, Representative fluorescence images showing SOX9 protein expression after AAV- shSox9 therapy in heart (scale bar, 1 mm) and brain (scale bar, 20 μm) of Ythdf3 +/+ and Ythdf3 -/- mice. d, Representative immunoblots showing SOX9 protein expression in heart tissues Ythdf3 +/+ and Ythdf3 -/- mice after AAV- shSox9 therapy. e , Echocardiographic analysis of Ythdf3 +/+ and Ythdf3 -/- mice after AAV- shSox9 therapy (n = 6). f, Quantitative PCR analysis of Sox9 downstream gene levels in heart tissues of Ythdf3 +/+ and Ythdf3 -/- mice after AAV- shSox9 therapy (n = 8). g, Representative images of Masson’s trichrome staining of heart sections of Ythdf3 +/+ and Ythdf3 -/- mice after AAV- shSox9 therapy. Scale bar, 200 μm. h, Heatmap of activity (left) and time spent in target quadrant (right) of Ythdf3 +/+ and Ythdf3 -/- mice given AAV- shSox9 therapy in the Morris water maze 24 h after training (n = 6). i, Top: representative images of Nissl-stained brain sections from Ythdf3 +/+ and Ythdf3 -/- mice after AAV- shSox9 therapy. Bottom: representative images of periodic acid-Schiff (PAS)-stained brain sections from Ythdf3 +/+ and Ythdf3 -/- mice after AAV- shSox9 therapy. Scale bar, 200 μm. Data represent the means ± SD. P -values were calculated by two-tailed unpaired Student’s t -test.

Article Snippet: Ythdf3 -knockout (KO) alleles were created by CRISPR–Cas9-mediated genome editing in C57BL/6 mice by the transgenic animal services of Cyagen Biosciences (Suzhou, China).

Techniques: Injection, Real-time Polymerase Chain Reaction, Fluorescence, Expressing, Western Blot, Staining, Activity Assay, Two Tailed Test