Journal: Communications Biology

Article Title: Infection with novel duck reovirus induces stress granule and methylation-mediated host translational shutoff in Muscovy ducklings

doi: 10.1038/s42003-024-07259-2

Figure Lengend Snippet: a Binding protein profiles of σB compared to λA. Confident candidate proteins were adjusted by the following conditions: log 2 FC > 1 or <−1 and FDR < 0.05. b Metascape was used to analyze the function of these candidate proteins (Supplementary Data ) with p values less than 0.05. c IP-WB shows the substantial interaction of YTHDF1, YTHDF3, CAPRIN1, and G3BP1 pulled down by Flag (σB) compared to that pulled down by Flag (λA). IP immunoprecipitation, WB Western blot.

Article Snippet: The membranes were then blocked with 5% nonfat milk TBS for 90 min and incubated with primary antibodies against YTHDF1 (1:2,000, Cat No. 17479-1-AP, Proteintech), YTHDF3 (1:2,000, Cat No. 25537-1-AP, Proteintech), CAPRIN1 (1: 2,000, Cat No. 15112-1-AP, Proteintech), G3BP1 (1: 2,000), Flag (1: 1,500), β-lactamase (encoding ampicillin resistant protein)(1: 2,000, Cat No. ab12251, Abcam), β-Actin (1: 2,500, Cat No. 81115-1-RR, Proteintech), σB and λA (1: 1,500, both antibodies were lab-made from mouse serum) overnight at 4 °C.

Techniques: Binding Assay, Immunoprecipitation, Western Blot

Journal: Communications Biology

Article Title: Infection with novel duck reovirus induces stress granule and methylation-mediated host translational shutoff in Muscovy ducklings

doi: 10.1038/s42003-024-07259-2

Figure Lengend Snippet: ICC shows the colocalization of σB and G3BP1 ( a ) or YTHDF1 ( b ) compared to that of EV and λA in duck embryo fibroblast CCL-141 cells. Images are shown at 80 × 10 magnification. Scale bars, 20 μm. c – e GF-WB image showing the coexpression (highlighted by the red frame) of YTHDF1, YTHDF3, CAPRIN1, G3BP1, and σB within the same protein fraction ( d ) compared to that of EVs ( c ) and λA ( e ). The numbers marked are the samples collected in sequence. f , g ICC showed increased expression of CAPRIN1 and G3BP1 in the bursa of Fabricius in Muscovy ducklings infected with NDRV HN10 ( f ) compared to NC ( e ) in vivo. Images are shown at 4 × 10 (left) and 40 × 10 (right) magnification. Scale bars, 20 μm. h , i Immunogold-labeled TEM image showing the colocalization of G3BP1 (large dots) and YTHDF1 (small dots) in the bursa of Fabricius in Muscovy ducklings infected with NDRV HN10 ( h ) compared to those infected with NC ( g ) in vivo. Scale bars, 200 nm (left) and 10 nm (right). ICC immunocytochemistry, GF gel filtration, WB Western blot, TEM transmission immunoelectron microscopy, EV empty vector, NC negative control.

Article Snippet: The membranes were then blocked with 5% nonfat milk TBS for 90 min and incubated with primary antibodies against YTHDF1 (1:2,000, Cat No. 17479-1-AP, Proteintech), YTHDF3 (1:2,000, Cat No. 25537-1-AP, Proteintech), CAPRIN1 (1: 2,000, Cat No. 15112-1-AP, Proteintech), G3BP1 (1: 2,000), Flag (1: 1,500), β-lactamase (encoding ampicillin resistant protein)(1: 2,000, Cat No. ab12251, Abcam), β-Actin (1: 2,500, Cat No. 81115-1-RR, Proteintech), σB and λA (1: 1,500, both antibodies were lab-made from mouse serum) overnight at 4 °C.

Techniques: Sequencing, Expressing, Infection, In Vivo, Labeling, Immunocytochemistry, Filtration, Western Blot, Transmission Assay, Immuno-Electron Microscopy, Plasmid Preparation, Negative Control

Journal: Communications Biology

Article Title: Infection with novel duck reovirus induces stress granule and methylation-mediated host translational shutoff in Muscovy ducklings

doi: 10.1038/s42003-024-07259-2

Figure Lengend Snippet: a WB was used to verify the efficiency of siRNA-mediated knockdown. b , c RIP-qPCR shows that the occupancy of Flag (σB) on the transcripts of Reg4 ( b ) and LOC113843712 ( c ) was reduced by YTHDF1/3 knockdown. d , e RIP-qPCR showed that the occupancy of G3BP1 on the transcripts of Reg4 ( d ) and LOC113843712 ( e ) was reduced by YTHDF1/3 knockdown. * indicates statistical significance compared to the NC group, with a P value less than 0.05 according to the student’s t-test. f IP-WB indicates that the substantial interaction between G3BP1 and CAPRIN1 was affected by both YTHDF1 and YTHDF3 knockdown. g The translational efficiency of the Muscovy duckling genes was reduced by YTHDF1/3 knockdown. A significant difference in the TEI between YTHDF1 knockdown (green) or YTHDF3 knockdown (purple) and NDRV HN10 knockdown (yellow) cells is indicated (YTHDF1, p = 2.8 × 10 −5 ; YTHDF3, p = 1.9 × 10 −6 ) by a two-tailed Wilcoxon rank-sum test. NC negative control, KD knockdown by siRNA, 1 KD YTHDF1 knockdown by siRNA, 3 KD YTHDF3 knockdown by siRNA, 1 and 3 KD both YTHDF1/3 knockdown by siRNA, TEI translational efficiency index.

Article Snippet: The membranes were then blocked with 5% nonfat milk TBS for 90 min and incubated with primary antibodies against YTHDF1 (1:2,000, Cat No. 17479-1-AP, Proteintech), YTHDF3 (1:2,000, Cat No. 25537-1-AP, Proteintech), CAPRIN1 (1: 2,000, Cat No. 15112-1-AP, Proteintech), G3BP1 (1: 2,000), Flag (1: 1,500), β-lactamase (encoding ampicillin resistant protein)(1: 2,000, Cat No. ab12251, Abcam), β-Actin (1: 2,500, Cat No. 81115-1-RR, Proteintech), σB and λA (1: 1,500, both antibodies were lab-made from mouse serum) overnight at 4 °C.

Techniques: Knockdown, Two Tailed Test, Negative Control

Journal: Nature Communications

Article Title: Autophagy induction promoted by m 6 A reader YTHDF3 through translation upregulation of FOXO3 mRNA

doi: 10.1038/s41467-022-32963-0

Figure Lengend Snippet: a Volcano plot depicting the intracellular proteins, detected by LC-MS/MS, that are differentially expressed in MEFs under nutrient-deprived versus normal conditions. The fold change values were calculated by dividing LFQ intensities measured in starved MEFs by intensities measured in normal MEFs. The significance threshold is set at a P- value below 0.05, from two-tailed unpaired t -tests performed on three biological replicates. Significantly up- and down-regulated proteins are denoted by red and blue dots, respectively. b Immunoblot analyses of YTH domain-containing proteins in MEFs in response to nutrient deficiency analyzed after the indicated time periods. β-actin is shown as a loading control. c Immunoblot analyses of LC3-II and p62, in shNS and two independent shYTHDF3 MEFs, following nutrient starvation for the indicated time periods, with and without Baf.A1 treatment (20 nM). GAPDH is used as a loading control. d Immunoblot analyses of YTHDF3 restored (shYTHDF3 + YTHDF3) and control (shYTHDF3 + Con) MEFs following nutrient starvation for the indicated time periods, with and without Baf.A1 treatment (20 nM). GAPDH is used as a loading control. e , f Representative confocal images of GFP-LC3 puncta formation ( e ) and quantification of GFP-LC3 puncta per cell ( f ) in shNS and shYTHDF3 MEFs. Nuclei were counterstained with DAPI. Scale bar, 20 μm. g , h Representative confocal images of GFP-LC3 puncta formation ( g ) and quantification of GFP-LC3 puncta per cell ( h ) in YTHDF3 restored and control MEFs. Nuclei were counterstained with DAPI. Scale bar, 20 μm. i Immunoblot analyses of LC3-II and p62 in control and YTHDF3 overexpressing MEFs, following nutrient starvation for the indicated time periods, with and without Baf.A1 treatment (20 nM). GAPDH is used as a loading control. j , k Representative confocal images of GFP-LC3 puncta formation ( j ) and quantification of GFP-LC3 puncta per cell ( k ) in control and YTHDF3 overexpressing MEFs. Nuclei were counterstained with DAPI. Scale bar, 20 μm. l Representative TEM images of autophagosomes (yellow arrow) and autolysosomes (red arrows) in wild-type and YTHDF3 −/− MEFs, with and without nutrient starvation. High magnification images of the boxed areas are displayed on the right-hand side. Scale bar, 1 μm. For f , h , k , mean numbers of puncta per cell from each randomly selected fields over three independent experiments were plotted (dots). Bars represent mean ± SEM. Two-tailed unpaired multiple t -tests with two-stage step-up correction (Benjamini, Krieger, and Yekutieli) were used to estimate significance. P -values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) constructs for mouse YTHDF3 and METTL3 were obtained according to the pLKO.1-puro vector protocol (Addgene, Cambridge, MA, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Western Blot, Control

Journal: Nature Communications

Article Title: Autophagy induction promoted by m 6 A reader YTHDF3 through translation upregulation of FOXO3 mRNA

doi: 10.1038/s41467-022-32963-0

Figure Lengend Snippet: a Representative confocal images of YTHDF3 and m 6 A fluorescence localization were obtained in MEFs following nutrient starvation for the indicated time periods. Nuclei were stained with DAPI. Scale bar, 20 μm. b Quantification of GFP-LC3 puncta per cell. c LC-MS/MS quantification of m 6 A levels in mRNA extracts from MEFs following nutrient starvation for the indicated time periods ( n = 3 biological replicates). d Immunoblot analyses of nuclear fractions from MEFs following nutrient starvation for the indicated time periods. e LC-MS/MS quantification of m 6 A levels in mRNA extracts from shNS and shMETTL3 MEFs, with or without nutrient deprivation ( n = 3 biological replicates). f The relative m 6 A methylation catalytic activities of purified METTL3 from the MEFs starved for the indicated time periods were determined using an RNA probe and d 3 -m 6 A. The methylation yields were calculated based on the molar ratio of newly formed d 3 -m 6 A to digested RNA probes ( n = 4, 4, 4, 2 biological replicates). G was used as an internal control to calculate the amount of RNA probes. g Immunoblot analysis of METTL3 in MEFs following nutrient starvation for the indicated time periods. h Immunoblot analyses of LC3-II and p62 in YTHDF3-OE MEFs infected with shNS or two independent METTL3 shRNAs (shMETTL3 and shMETTL3-2) following nutrient starvation for the indicated time periods, with or without Baf.A1 treatment (20 nM). GAPDH was used as a loading control. i Immunoblot analyses of METTL3-silenced YTHDF3-OE MEFs transfected with lentiviral vectors (Con), wild-type METTL3 (METTL3-WT), or a catalytic mutant of METTL3 (METTL3-Mut) following nutrient starvation for the indicated time periods, with or without Baf.A1 treatment (20 nM). GAPDH is used as a loading control. j , k Measurement of autophagy flux and quantification of autophagosomes (yellow) and autolysosomes (red) by a tandem mCherry-GFP-LC3 reporter assay in shNS and shMETTL3 MEFs of YTHDF3-OE, with or without nutrient deficiency. Scale bar, 20 μm. l , m Measurement of autophagy flux and quantification of autophagosomes (yellow) and autolysosomes (red) by a tandem mCherry-GFP-LC3 reporter assay in METTL3-silenced YTHDF3-OE MEFs transfected with METTL3-WT, METTL3-Mut, or Con, with or without nutrient deficiency. Scale bar, 20 μm. For b , k , m mean numbers of puncta per cell from each randomly selected fields over three independent experiments were plotted (dots). All bars represent mean ± SEM. Two-tailed unpaired t -tests ( b , c , e , f ), or two-tailed unpaired multiple t -tests with two-stage step-up correction (Benjamini, Krieger, and Yekutieli) ( k , m ), were used to estimate the significance. P -values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) constructs for mouse YTHDF3 and METTL3 were obtained according to the pLKO.1-puro vector protocol (Addgene, Cambridge, MA, USA).

Techniques: Fluorescence, Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Methylation, Purification, Control, Infection, Transfection, Mutagenesis, Reporter Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Autophagy induction promoted by m 6 A reader YTHDF3 through translation upregulation of FOXO3 mRNA

doi: 10.1038/s41467-022-32963-0

Figure Lengend Snippet: a The scatter plot depicts fold changes (log2) of YTHDF3-RIP target peaks in MEFs after nutrient deprivation. Red dots indicate significantly up-enriched and down-enriched peaks with a cutoff fold change of 1.8. b The consensus sequence motif identified within significant differentially enriched YTHDF3-binding sites, determined by the HOMER database. c Metagene profiles of the significant differentially enriched YTHDF3-binding sites along a normalized transcript, consisting of three rescaled non-overlapping segments: 5’UTR, CDS, and 3’UTR. Pie chart depicting the fraction of significant differentially enriched YTHDF3-binding sites in different transcript segments. d Scatter plot showing m 6 A peaks with increased (red) or decreased (blue) levels in response to nutrient deficiency. e Venn diagram showing the number of overlapping up-enriched YTHDF3-binding targets and hyper-m 6 A-methylated mRNAs upon nutrient deprivation. Then the resultant 86 peaks were annotated to GO term autophagy (0006914) and obtained 7 genes. f Integrative Genomics Viewer (IGV) tracks displaying YTHDF3-RIP-seq (upper panels) and MeRIP-seq (lower panels) read distribution along the CDS and 3’UTR of FOXO3 mRNA. The squares mark increases in m 6 A peaks in MEFs upon nutrient deficiency. g , h Gene-specific MeRIP-qPCR analysis of m 6 A level changes at the CDS ( g ) and 3’UTR ( h ) regions of FOXO3 mRNA transcripts in shNS and shMETTL3 MEFs upon nutrient deficiency ( n = 3 biological replicates). i YTHDF3-RIP followed by qRT-PCR confirmed the interaction between YTHDF3 and FOXO3 mRNA in shNS and shMETTL3 MEFs upon nutrient deficiency ( n = 3 biological replicates). j , k RNA EMSA assays were performed using recombinant YTHDF3 proteins and biotinylated RNA probes containing different sequences of FOXO3’s CDS ( j ) or 3’UTR ( k ) with or without m 6 A modifications. Data are presented as mean values ± SEM. Two-tailed unpaired t -tests were used to estimate significance. P -values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) constructs for mouse YTHDF3 and METTL3 were obtained according to the pLKO.1-puro vector protocol (Addgene, Cambridge, MA, USA).

Techniques: Sequencing, Binding Assay, Methylation, Quantitative RT-PCR, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Autophagy induction promoted by m 6 A reader YTHDF3 through translation upregulation of FOXO3 mRNA

doi: 10.1038/s41467-022-32963-0

Figure Lengend Snippet: a Immunoblot analyses of FOXO3 and p-FOXO3 in shNS, shYTHDF3, control, and YTHDF3-OE MEFs, with or without nutrient starvation, respectively. b qRT-PCR analysis of mRNA levels of FOXO3 target autophagy-related genes in shNS, shYTHDF3, control, and YTHDF3-OE MEFs ( n = 3 biological replicates). c Immunoblot analyses of protein levels of FOXO3-targeted autophagic genes in shNS, shYTHDF3, control, and YTHDF3-OE MEFs. d Immunoblot analyses of nuclear and cytoplasmic FOXO3 expressions in METTL3-silenced YTHDF3-OE MEFs and control MEFs following nutrient starvation for the indicated time periods. e Immunoblot analyses of protein levels of FOXO3-targeted autophagic genes in shNS, shMETTL3, control, and METTL3-OE MEFs. f qRT-PCR analysis of mRNA levels of FOXO3 target autophagy-related genes in METTL3-silenced YTHDF3-OE MEFs and control MEFs ( n = 3 biological replicates). g Immunoblot analyses of nuclear and cytoplasmic FOXO3 in FOXO3 rescued MEFs (shYTHDF3 + FOXO3) and control MEFs (shYTHDF3 + Con). GAPDH is used as a loading control. h Immunoblot analyses of FOXO3 rescued MEFs (shYTHDF3 + FOXO3) and control MEFs (shYTHDF3 + Con) following nutrient starvation for the indicated time periods, with or without Baf.A1 treatment (20 nM). i qRT-PCR analysis of mRNA levels of FOXO3 target autophagy-related genes in FOXO3 rescued and control MEFs ( n = 3 biological replicates). j Immunoblot analyses of protein levels of FOXO3 targeted autophagic genes in FOXO3 rescued MEFs and control MEFs. GAPDH is used as a loading control. k , l mCherry-GFP-LC3 was transfected into FOXO3 rescued and control MEFs, and the formation of autophagosomes (yellow) and autolysosomes (red) was examined. Scale bar, 20 μm. m Immunoblot analyses of FOXO3-silenced YTHDF3-OE MEFs (YTHDF3 + siFOXO3) and control MEFs (YTHDF3 + siNC) following nutrient starvation for the indicated time periods, with or without Baf.A1 treatment (20 nM). n , o mCherry-GFP-LC3 was transfected into FOXO3-silenced YTHDF3-OE MEFs and control MEFs, and the formation of autophagosomes (yellow) and autolysosomes (red) was examined. Scale bar, 20 μm. For i , o , mean numbers of puncta per cell from each randomly selected fields over three independent experiments were plotted (dots). All bars represent mean ± SEM. Two-tailed unpaired multiple t -tests with two-stage step-up correction (Benjamini, Krieger, and Yekutieli) were used to estimate significance. P -values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) constructs for mouse YTHDF3 and METTL3 were obtained according to the pLKO.1-puro vector protocol (Addgene, Cambridge, MA, USA).

Techniques: Western Blot, Control, Quantitative RT-PCR, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Autophagy induction promoted by m 6 A reader YTHDF3 through translation upregulation of FOXO3 mRNA

doi: 10.1038/s41467-022-32963-0

Figure Lengend Snippet: a qRT-PCR analysis of FOXO3 in shNS and shYTHDF3 MEFs following nutrient starvation for the indicated time periods. b Sucrose gradient-based polysome profiling of shNS and shYTHDF3 MEFs. c FOXO3 mRNAs in each ribosome fraction were quantified through qRT-PCR and plotted as percentages of the total. d Lv-FOXO3-CDS-WT, Lv-FOXO3-CDS-Mut1, or Lv-FOXO3-CDS-Mut2 were transfected into control and YTHDF3-OE MEFs. Protein expression was measured by western blot analysis and then quantitatively analyzed. e Schematic diagram of wild-type or mutant m 6 A sites (A-to-T mutation) in the FOXO3-3’UTR, fused with a dual luciferase reporter. f FOXO3-3′UTR-WT or FOXO3-3’UTR-Mut reporters were transfected into control and YTHDF3-OE MEFs for 72 h. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. g After transfecting FOXO3-3′UTR-WT or FOXO3-3′ UTR-Mut, MEFs were nutrient starved. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. h shNS and shYTHDF3 MEFs were treated with Act.D (5 μg/mL) for the indicated times. The expression of FOXO3 was examined with qRT-PCR. Data from three independent experiments are expressed as mean values ± SEM. P -values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) constructs for mouse YTHDF3 and METTL3 were obtained according to the pLKO.1-puro vector protocol (Addgene, Cambridge, MA, USA).

Techniques: Quantitative RT-PCR, Transfection, Control, Expressing, Western Blot, Mutagenesis, Luciferase, Activity Assay

Journal: Nature Communications

Article Title: Autophagy induction promoted by m 6 A reader YTHDF3 through translation upregulation of FOXO3 mRNA

doi: 10.1038/s41467-022-32963-0

Figure Lengend Snippet: a YTHDF3 immunoprecipitation workflow in MEFs under normal and nutrient-free conditions. b YTHDF3-specific interactors were identified through quantitative mass spectrometry. eIFs are labeled in red. 40S and 60S ribosomal subunits are labeled in light blue. Diameters correlate with mean numbers of unique peptides ( n = 2). c Immunoblot analyses for eIF3a and eIF4B proteins from polysome fractions in shNS and shYTHDF3 MEFs. d Surface view of docked YTHDF3-eIF3a and YTHDF3-eIF4B complexes. YTHDF3, eIF3a, and eIF4B are colored in blue, green, and red, respectively. Inset, magnified views of the interacting residues are drawn in stick representation and labeled (in pink for YTHDF3 and in yellow for eIF3a or eIF4B). e Immunoblot analyses of YTHDF3-immunoprecipitated proteins from shNS and shYTHDF3 MEFs, with or without nutrient deprivation. Total protein amounts of YTHDF3, eIF3a, and eIF4B were used as inputs. f Immunoblot analyses of YTHDF3 immunoprecipitation lysates, with or without RNase A treatment. eIF3a and eIF4B were detected. g Immunoblot analyses of FOXO3 in sheIF3a, sheIF4B, and control MEFs, respectively. h Immunoblot analyses for YTHDF3 in PABP1- or eIF4G2-immunoprecipitated lysates from MEFs with or without nutrient deprivation. Total protein amounts of PABP1, eIF4G2, and YTHDF3 were used as inputs. Source data are provided as a Source Data file.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) constructs for mouse YTHDF3 and METTL3 were obtained according to the pLKO.1-puro vector protocol (Addgene, Cambridge, MA, USA).

Techniques: Immunoprecipitation, Mass Spectrometry, Labeling, Western Blot, Control

Journal: Nature Communications

Article Title: Autophagy induction promoted by m 6 A reader YTHDF3 through translation upregulation of FOXO3 mRNA

doi: 10.1038/s41467-022-32963-0

Figure Lengend Snippet: Under nutrient deprivation, the m 6 A reader YTHDF3 and m 6 A writer METTL3 are up-regulated. The latter promotes m 6 A hypermethylation at the CDS and 3’UTR regions around the stop codon of the FOXO3 transcript. YTHDF3 facilitates FOXO3 translation by binding to these m 6 A modifications around the stop codon and recruiting the eIF3a subunit and eIF4B to the 5’ end of FOXO3 mRNA, forming mRNA loops. FOXO3 then activates the transcription of core autophagy-related genes and promotes autophagy.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) constructs for mouse YTHDF3 and METTL3 were obtained according to the pLKO.1-puro vector protocol (Addgene, Cambridge, MA, USA).

Techniques: Binding Assay

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

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: Primer sequences used for RT-qPCR.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Marker

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: Identification of 8 AAA-related m1A regulators.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Quantitative Proteomics

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: The differential expression level and diagnostic value of DEMRGs in AAA. (A) The up-regulating expression level of YTHDF1 ; (B) The up-regulating expression level of YTHDF2 ; (C) The up-regulating expression level of YTHDF3 ; (D) The down-regulating expression level of FTO ; (E) The up-regulating expression level of YTHDC1 ; (F) The up-regulating expression level of RRP8 ; (G) The up-regulating expression level of TRMT61A ; (H) The down-regulating expression level of ALKBH1 ; (I) The LASSO regression model established by 8 DEMRGs; (J) The coefficients plot of 8 DEMRGs in the LASSO model; (K) The ROC curves with AUC values of 6 key AAA-related m1A regulators.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Quantitative Proteomics, Diagnostic Assay, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: The relationships between DEMRGs and immune cells. (A) Scatter plot of the correlation between YTHDF3 and activated mast cells; (B) Scatter plot of the correlation between YTHDF3 and plasma cells; (C) Scatter plot of the correlation between YTHDF3 and regulatory T cells (Tregs); (D) Scatter plot of the correlation between YTHDF3 and M1 macrophages; (E) Scatter plot of the correlation between YTHDF1 and M1 macrophages; (F) Scatter plot of the correlation between YTHDF2 and M1 macrophages; (G) Scatter plot of the correlation between YTHDF1 and M2 macrophages; (H) Scatter plot of the correlation between YTHDF3 and M2 macrophages; (I) Correlation heat map of the relationships between DEMRGs and immune cells, with red representing positive correlation, blue representing negative correlation and NA representing no statistical significance.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Clinical Proteomics

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: The validation of the expression of DEMRGs at mRNA level in AAA tissue samples compared with the healthy control aortic samples, analyzed by RT-qPCR. (A) The relative expression of YTHDC1 in two groups; (B) The relative expression of YTHDF1 in two groups; (C) The relative expression of YTHDF3 in two groups; (D) The relative expression of YTHDF2 in two groups; (E) The relative expression of RRP8 in two groups; (F) The relative expression of TRMT61A in two groups. * P < 0.05.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Biomarker Discovery, Expressing, Control, Quantitative RT-PCR

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: The expression of YTHDF3 at protein level and its cellular localization in AAA tissues. (A) The images of Western Blot staining for YTHDF3 and GAPDH in each sample; (B) The box plot showing the relative expression of YTHDF3 (normalized to the signal intensity of GAPDH ) in AAA and normal aortic samples; (C) The photographs of immunofluorescence for human AAA sections stained with CD68, YTHDF3 and 4', 6-diamidino-2-phenylindole (DAPI). * P < 0.05.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: The induction of macrophages M1 polarization and the determination of YTHDF3 expression in M0 and M1 macrophages. (A) The expression of M1 phenotype marker, CD86 , in M0 macrophages and LPS/IFN-γ induced M1 macrophages, measured by RT-qPCR; (B) The expression of M1 phenotype marker, IL12, in M0 macrophages and LPS/IFN-γ induced M1 macrophages, measured by ELISA; (C) The images of Western Blot staining for YTHDF3 and β -Actin , in M0 and LPS/IFN-γ induced M1 macrophages; (D) The relative expression of YTHDF3 (normalized to the signal intensity of β -Actin ) in M0 and LPS/IFN-γ induced M1 macrophages. LPS, lipopolysaccharide; IFN-γ, interferon-γ. ** P < 0.01, *** P < 0.001.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Expressing, Marker, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: The role of YTHDF3 in the M1/M2 polarization of M0 macrophages. YTHDF3 ; (A) The relative expression of M1 phenotype markers ( CD86, iNOS and TNF α) in M0 macrophages and si- ythdf3 macrophages, analyzed by RT-qPCR; (B) The relative expression of M2 phenotype markers ( CD206, Arg-1 and TGF β) in M0 macrophages and si- ythdf3 macrophages, analyzed by RT-qPCR; (C) The images of Western Blot staining for iNOS and β -Actin , in M0 macrophages, LPS/IFN-γ induced M1 macrophages and si- ythdf3 +M1 polarization macrophages; (D) The relative expression of iNOS (normalized to the signal intensity of β -Actin ) in M0 macrophages, LPS/IFN-γ induced M1 macrophages and si- ythdf3 +M1 polarization macrophages; (E) The expression of IL12 in M0 macrophages, LPS/IFN-γ induced M1 macrophages and si- ythdf3 +M1 polarization macrophages, measured by ELISA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; iNOS , inducible nitric oxide synthase; TNF α, tumor necrosis factor-α; Arg-1 , arginine-1; TGF β, transforming growth factor-β.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization

doi: 10.3389/fcvm.2022.883155

Figure Lengend Snippet: The construction of PPI network based on the RIP-Seq result and the prediction of YTHDF3 downstream targets participating in AAA progression. (A) The Venn diagram showing the overlapping of the AAA-specific YTHDF3 -binding genes YTHDF3, YTHDF3 co-expressed (+) genes and up-regulated DEGs in AAA. (B) The two-level PPI network centered on YTHDF3 ; (C) The interactions among proteins with the top 20 MCC values in the whole PPI network. Red represents higher MCC values and yellow represents lower MCC values; (D) The interactions among proteins with the top 20 node degrees in the whole PPI network. Red represents higher node degrees and yellow represents lower node degrees; (E) The sub-network with the highest cluster score in the whole PPI network. Hub genes (identified by MCC method) existing in this sub-network were marked yellow; (F) The sub-network with the highest cluster score in the whole PPI network. Hub genes (identified by Degree method) existing in this sub-network were marked yellow. MCC, Maximal Clique Centrality.

Article Snippet: Small interfering RNA (siRNA) against mouse YTHDF3 as well as Negative Control (NC) siRNA was designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Binding Assay

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

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