Journal: PLoS Pathogens

Article Title: Norovirus infection results in eIF2α independent host translation shut-off and remodels the G3BP1 interactome evading stress granule formation

doi: 10.1371/journal.ppat.1008250

Figure Lengend Snippet: G3BP1 interactome differs in MNV-infected cells and arsenite treated cells as addressed by immunofluorescence (A-C). BV2 GFP-G3BP1 cells were either infected with MNV (MOI 10) for 9h p.i. or treated with arsenite 0.1mM for 1h and subcellular colocalisation of candidate proteins identified by mass spectrometry as interacting with GFP-G3BP1 was addressed by immunostaining (magenta) in GFP-G3BP1 positive cells (cyan). MNV-infected cells were detected by immunostaining against dsRNA (gold), nuclei were stained with DAPI. Scale bars, 10μm, red arrows indicate example of colocalisation between GFP-G3BP1 and a candidate protein. Representative view (n = 3) of confocal analysis using staining against (A) YTHDF3, candidate for arsenite-induced SG only, (B) FMR1, candidate for both arsenite- and MNV-induced G3BP1 granules, (C) Rab7, candidate for MNV-induced G3BP1 granules only.

Article Snippet: Primary antibodies dilutions: rabbit anti-NS3 (1:600), mouse anti-dsRNA (1:1,000), mouse anti-puromycin (1:5, http://dshb.biology.uiowa.edu/PMY-2A4 ), mouse anti-G3BP1 (1:400, Invitrogen), goat anti-eIF3B (1:400, Santa Cruz), rabbit YTHDF3 (1:600, Proteintech), rabbit anti-RAB7A (1:600, Proteintech), rabbit anti-FMR1 (1:600, Novusbio).

Techniques: Infection, Immunofluorescence, Mass Spectrometry, Immunostaining, Staining

Journal: BMC Cancer

Article Title: N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma

doi: 10.1186/s12885-022-10010-x

Figure Lengend Snippet: Summary of the analyzed m6A proteins as indicated and their correlation with overall survival (indicated as %alive) for the entire study cohort, HPV-independent, and HPV-dependent VSCC. The HPV-status was not available for 24 patients. Samples were grouped according to high and low expression based on the staining intensities. p -values for the group comparisons are based on log-rank tests (significance threshold p < 0.5). q -values are based on multiple hypotheses testing using the method of Benjamini and Hochberg with a significance threshold of q < 0.1

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on the TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82,188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4 °C overnight.

Techniques: Expressing, Staining

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

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202301538

Figure Lengend Snippet: Figure 4. 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

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955-1-AP), Electroporation: Denuded mouse GV oocytes were collected as described above.

Techniques: In Vitro, Control, Two Tailed Test, MANN-WHITNEY, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202301538

Figure Lengend Snippet: Figure 5. 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

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955-1-AP), Electroporation: Denuded mouse GV oocytes were collected as described above.

Techniques: Binding Assay