ythdf3 knockout ko alleles  (Cyagen Biosciences)

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