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: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Mettl3 is essential for female mice fertility. (A). Phylogenetic tree of the protein sequences of Ythdf1, Ythdf2 and Ythdf3, based on UCSC database. The three readers appeared together in vertebrates, possibly after whole genome duplication. (B) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in ICR wild-type (WT) oocytes. (BF) Bright field. (C) H&E staining of ovaries, showing normal follicle structure in Mettl3f/f VasaCre− control, and a severe abnormality in Mettl3f/f VasaCre+ females. (D) Gross morphology of Mettl3f/fVasaCre+ and control female ovaries. Cre+ females show a smooth shape that lacks the typical follicular morphology. (E) Number of pups per plug produced by mating Mettl3f/f Vasa Cre+ females, compared with Mettl3f/f Vasa Cre− control females. The fathers in both cases were WT. A significant difference between Cre+ and Cre− female fertility is observed (P < 0.0001, Mann–Whitney test). (F) H&E staining of ovaries, showing normal morphology both in Mettl3f/f Zp3 Cre− and Mettl3f/f Zp3 Cre+ ovaries. (G) Number of pups per plug produced by mating a Mettl3f/f Zp3 Cre+ female, compared with a Mettl3f/+ Zp3 Cre+ control female. The fathers in both cases were WT. A significant difference between f/f and f/+ female fertility is observed (P < 0.0001, Mann-Whitney test). (H) Number of oocytes per mouse flushed from Mettl3f/f Zp3 Cre+ females, compared with Mettl3f/f Zp3 Cre− control females. A significant difference between the number of oocytes of Mettl3f/f ZP3 Cre+ and Mettl3f/f ZP3 Cre− is observed (P < 0.0002, Mann–Whitney test). (I, top) Experimental design – Mett3f/f Zp3 Cre+ and Cre− as control underwent hormone priming, oocyte flushing, fixation, and staining for tubulin. (Bottom) Number of oocytes observed in the different stages of meiosis. In the control, most of the oocytes were in MI stage, in KO (Cre+) all of the observed oocytes were in the GV state. (J) Staining examples of oocytes in the different stages of meiosis as observed in KO (Cre+) and control (Cre−). (K) Differentially expressed genes between Mettl3f/f Zp3 Cre− (control) and Mettl3f/f Zp3 Cre+ (KO) oocytes, along with selected enriched categories. m6A methylated genes appear in bold. Ninety-six genes are up-regulated in the KO, and 117 are down-regulated in the KO.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Immunostaining, Staining, Produced, MANN-WHITNEY, Methylation

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Characterization of compound Ythdf1/2/3 KO mice. (A, left) Statistics of KO offspring received from crossing of Heterozygous mice from each of the indicated strains (Ythdf1+/−, Ythdf2+/−, and Ythdf3+/−). (Right) Distribution of Ythdf2 WT, HET, and KO offspring at days E13.5, 2 d postnatal (DPN), and 30 DPN (compared with expected ratios). (B) Crossing strategy for generating triple-heterozygous mice that were further crossed, and their offspring statistics are presented in C. (C) Percentage of genotypes received by crossing triple-heterozygous mice, out of 200 pups tested 30 DPN. (Red) Observed percentage; (gray) expected under null assumption. No pups with Ythdf2-KO genotype survived 30 DPN. In Ythdf2+/− genotype, pups with KO in either Ythdf1 or Ythdf3 were born in a sub-Mendelian ratio. χ2 test P-values are indicated. (D) Using tetraploid complementation assay, triple-KO embryos were generated and examined on E7.5. (Bottom) H&E staining showing aberrant morphology of triple-KO E7.5 embryos, compared with WT control.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Generated, Staining

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf2 is essential for male mice fertility. (A) H&E staining showing mild degenerative changes, including scattered vacuoles marked by arrows in the seminiferous tubules in Ythdf2-KO males, compared with WT control. (B) Schematic representation of spermatogenesis inside seminiferous tubules. Differentiation is progressing from spermatogonia at the periphery, via spermatocytes and round spermatids, and ends with elongated mature sperm in the center of the tubule. (C, left) H&E staining of the cauda epididymis, showing severe loss of sperm in Ythdf2-KO compared with control. (Right) Bright field of sperm extracted from the cauda epididymis of KO and control, showing a severe reduction in normal sperm quantity in the KO sample, compared with control. (D) Number of pups per plug produced by mating Ythdf2−/− males, compared with Ythdf2+/− control males. The mothers in both cases were WT. A significant difference between the fertility of KO and heterozygous males is observed (P < 0.0001, Mann–Whitney test). (E) Immunostaining of Ythdf1, Ythdf2, Ythdf3, and Mettl3 in seminiferous tubules, showing that each of the Ythdf proteins is expressed at different stages of spermatogenesis. Costaining of Gata4 typically marks Sertoli cells, costaining of γ-H2AX marks different cells during early spermatogenesis. (F) Dimension reduction representation of single-cell RNA-seq (UMAP) measured in adult mouse testis, showing mild expression of Ythdf1 and Ythdf3 in spermatogonia and in Sertoli cells, and more substantial expression of Ythdf2 in spermatogonia and in spermatocytes.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Staining, Produced, MANN-WHITNEY, Immunostaining, RNA Sequencing Assay, Expressing

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf1, Ythdf2, and Ythdf3 are redundant in mouse naïve ESC maintenance. (A) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in WT mESCs, showing a protein expression in the cytosolic compartment of the cell. (B) Cell growth curve of all KO lines and WT control. Cells were grown on mouse feeders, in serum/LIF conditions. (C) Bright field and immunostaining of Nanog (green), Esrrb (red), and DAPI (blue) in KO cells (single, triple, and Mettl3) and WT control, showing that all cell lines express Nanog and Esrrb.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Immunostaining, Expressing

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf1, Ythdf2, and Ythdf3 are functionally redundant in ESC differentiation. (A) Teratomas generated from KO cell line and from WT control. Single-KO teratomas show all germ layers, while triple-KO (TKO) teratomas are poorly differentiated. Selected differentiated structures are marked by arrows. (B) Immunostaining of triple-KO and WT control to Oct4 (red), Foxa2 (green), Tuj1(purple) and DAPI (blue). Triple-KO teratomas contain patches of Oct4 staining, unlike WT teratomas. (C) Alkaline phosphatase (AP) staining of disassociated teratomas from triple-KO and WT control samples, showing a greater AP staining in the Ythdf triple-KO. (D) RT-PCR of pluripotency genes (left) and differentiation genes (right), measured in WT mESCs, and mEBs from the following cell lines: WT control, Ythdf single-KO, Ythdf triple-KO, and Ythdf triple-KO + overexpression (OE) of Ythdf1/2/3 (rescued cell lines). Triple-KO EBs express pluripotent markers and repress differentiation markers similarly to mESCs. Rescued EBs express differentiation markers, similarly to single-KO EBs. (E) EBs were induced for 7 d from the indicated cell lines, and dissociated into single cells at day 7 and comparable amounts of cells were replated in ES medium. Undifferentiated AP+ colonies number per 1000 plated cells was evaluated 5 d later.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Generated, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction, Over Expression

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf triple-KO has a dramatic effect on gene expression. (A) Hierarchical clustering of samples based on Pearson correlation, showing that single-KO samples are highly similar to WT. (B) Number of differentially expressed genes in each of the KO cell lines, compared with WT. (Black) Down-regulated genes, (gray) up-regulated genes. (C) RNA-seq and m6A methylation landscape of selected genes. Normalized coverage is presented. Only Nanog and Dnmt3l are m6A methylated. Dnmt3l, Zscan4a, Zscan4b, Zscan4d, and Dppa3 are overexpressed in triple-KO. (D) Enrichment of up-regulated genes in each category, to early embryo genes (Gao et al. 2017). Genes that are up-regulated in KO of Ythdf1 and Ythdf3 are specifically enriched for two-cell embryo genes. (E) Normalized expression of Ythdf1, Ythdf2, and Ythdf3, as measured in early mouse embryo (Gao et al. 2017). (F) CLIP coverage over m6A peaks of Ythdf readers as measured in humans (n = 41,885) (Patil et al. 2016), and in mice (n = 9861), showing high correlation between coverage by different Ythdf readers.

Article Snippet: Generation of Ythdf1, Ythdf2, and Ythdf3 knockout murine ESC lines with CRISPR/Cas9 To knock out the Ythdf genes in mESCs, oligos for gRNAs were cloned into px335 vector (Addgene 42335) encoding SpCas9 nickase.

Techniques: Expressing, RNA Sequencing Assay, Methylation

Journal: Genes & Diseases

Article Title: YTHDF1 shapes immune-mediated hepatitis via regulating inflammatory cell recruitment and response

doi: 10.1016/j.gendis.2024.101327

Figure Lengend Snippet: The protein level of m 6 A reader YTHDF1, YTHDF2 and YTHDF3 decrease during the early stage of ConA-induced hepatitis. Eight-week-old male C57BL/6 WT mice were intravenously injected with ConA (10 mg/kg) or saline as vehicle control, and the liver tissues were collected at various time points after ConA treatment, n = 2–3 per group. ( A, B ) Immunoblot analysis of liver tissues with m 6 A family and representative inflammatory pathway antibodies (A), followed by gray analysis using Image J (B). (C) Representative images of liver sections for immunostaining analysis with YTHDF1, YTHDF2 and YTHDF3 antibodies, Scale bar = 100 μm. (D) Heatmap analysis from liver RNA-seq data indicated the mRNA expression level of the m 6 A family after ConA treatment for 8 h. (E, F) Heatmap analysis from the GEO database showed the fold change of m 6 A family mRNA levels after the ConA challenge at indicated time points compared with vehicle control in liver tissues. Data are presented as mean ± SD; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared to the control group of WT ConA 0h, based on unpaired two-sided Student's t -test.

Article Snippet: C57BL/6 YTHDF1 knockout ( Ythdf1 −/− ) mice, YTHDF3 knockout (Ythdf3 −/−) mice, and Rosa26-LSL-Cas9-tdTomato ( Cas9 +/− ) mice were obtained from Cyagen Bioscience and Gempharmatech Inc. Wild type (WT), Ythdf1 −/− , Ythdf3 −/− , and Cas9 +/− mice on C57BL/6 background were bred and housed in specific pathogen-free conditions at the Xiamen University Laboratory Animal Center.

Techniques: Injection, Saline, Control, Western Blot, Immunostaining, RNA Sequencing, Expressing

Journal: Genes & Diseases

Article Title: YTHDF1 shapes immune-mediated hepatitis via regulating inflammatory cell recruitment and response

doi: 10.1016/j.gendis.2024.101327

Figure Lengend Snippet: Deletion of YTHDF1, not YTHDF3, exacerbates ConA-induced liver injury and mortality in mice. (A) The mortality rate of YTHDF1 knockout ( Ythdf1 −/− ) and wildtype littermates (WT) male mice at various time points after receiving an intravenous lethal dose of ConA (20 mg/kg), n = 15–17 per group. (B – E) Ythdf1 −/− and WT male mice were administrated with ConA (8 mg/kg) or saline by intravenous injection, n = 3–5 per group. Serum samples or liver tissues were collected at 0 h, 3 h, 8 h, and 24 h after ConA injection. Measurement of Serum ALT (B) and AST (C) levels. H&E staining analysis of liver sections, Scale bar = 200 μm (D), the necrotic area was analyzed by image J (E). (F) The mortality rate of Ythdf3 −/− and WT male mice at various time points after receiving an intravenous lethal dose of ConA (20 mg/kg), n = 15–17 per group. (G, H) Ythdf3 −/− and WT male mice were intravenously injected with ConA (8 mg/kg) or saline for 8 h, and serum samples were collected for the measurement of ALT (G) and AST (H) levels, n = 3–5 per group. Data are presented as mean ± SD; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant, compared with two groups of the corresponding time-point, based on two-sided Student's t -test. The survival curves are analyzed by the Log-rank test.

Article Snippet: C57BL/6 YTHDF1 knockout ( Ythdf1 −/− ) mice, YTHDF3 knockout (Ythdf3 −/−) mice, and Rosa26-LSL-Cas9-tdTomato ( Cas9 +/− ) mice were obtained from Cyagen Bioscience and Gempharmatech Inc. Wild type (WT), Ythdf1 −/− , Ythdf3 −/− , and Cas9 +/− mice on C57BL/6 background were bred and housed in specific pathogen-free conditions at the Xiamen University Laboratory Animal Center.

Techniques: Knock-Out, Saline, Injection, Staining