human ythdf1  (OriGene)

Journal: Science Advances

Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response

doi: 10.1126/sciadv.ado7660

Figure Lengend Snippet: ( A ) Immunofluorescence detection of YTHDF1 using anti-YTHDF1 in HEK293T cells with or without x-ray treatment (a dose of 4 Gy). Nuclei were counterstained with Hoechst (blue). Scale bars, 10 μm. CTL, control. DAPI, 4′,6-diamidino-2-phenylindole. ( B ) Immunoblotting analysis of total, cytosolic (Cyto), and nuclear (Nuc) fractions of HEK293T cells with or without x-ray treatment (a dose of 4 Gy). ( C ) Immunoblotting analysis confirming the efficiency of ATR and ATM inhibitors (ATRi and ATMi). HEK293T cells treated with KU60019 (10 μM) or VE-821 (10 μM) for 24 hours and x-ray were subjected to immunoblotting analysis. ( D ) Immunoblotting analysis confirming the efficiency of DNA-PKcs inhibitor (DNA-PKcsi). HEK293T cells treated with NU7441 (2 μM) for 24 hours and x-ray were subjected to immunoblotting analysis. ( E ) Subcellular localization of FLAG-tagged YTHDF1 under the same conditions as (C) and (D). Lentiviral transduction was used for the expression of YTHDF1-FLAG. ( F ) Validation of ATR knockdown efficiency by immunoblotting in HEK293T cells. ( G ) Intracellular localization of YTHDF1 in control and ATR knockdown cells with x-ray treatment (a dose of 4 Gy). ( H ) Coimmunoprecipitation experiment confirming the interaction between ATR and YTHDF1. Lysates from HEK293T cells with or without x-ray treatment (a dose of 4 Gy) were immunoprecipitated with immunoglobulin G (IgG) or anti-ATR and then immunoblotted with anti-YTHDF1 antibody. IP, immunoprecipitation. ( I ) Intracellular localization of alanine mutants of YTHDF1 in HEK293T cells. A dose of 4-Gy x-ray irradiation was applied. ( J ) In vitro kinase assay using antibody-affinity–isolated ATR complex with purified YTHDF1 or YTHDF1-S182A. The phosphorylation was detected using a p-serine antibody (top). YTHDF1 and ATR were detected by Western blot (middle) and Coomassie blue staining (bottom). GFP, green fluorescent protein.

Article Snippet: To achieve YTHDF1 ablation, single-guide RNA targeting human YTHDF1 was subcloned into the vector lentiCRISPR v2 (52961, Addgene) at the Bsm BI site.

Techniques: Immunofluorescence, Control, Western Blot, Transduction, Expressing, Biomarker Discovery, Knockdown, Immunoprecipitation, Irradiation, In Vitro, Kinase Assay, Isolation, Purification, Phospho-proteomics, Staining

Journal: Science Advances

Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response

doi: 10.1126/sciadv.ado7660

Figure Lengend Snippet: ( A ) The consensus NES of YTHDF1 in representative species. The hydrophobic residues are shown within red frames, while the phosphorylation site is shown within a blue frame. ( B ) Subcellular localization of FLAG-tagged YTHDF1 mutants in HEK293T cells. Nuclei were counterstained with Hoechst (blue). Scale bars, 10 μm. ( C ) Coimmunoprecipitation analysis of CRM1 interaction with YTHDF1. Lysates from HEK293T cells expressing FLAG-CRM1 were subjected to immunoprecipitation using either IgG or anti-FLAG antibody, followed by immunoblotting with an anti-YTHDF1 antibody. ( D ) Intracellular localization of endogenous YTHDF1 in HEK293T cells with or without 1-hour LMB treatment. Scale bars, 10 μm. ( E ) Coimmunoprecipitation analysis of FLAG-CRM1 interaction with YTHDF1 with or without x-ray treatment (a dose of 4 Gy). ( F ) Coimmunoprecipitation analysis of endogenous CRM1 interaction with YTHDF1 with or without x-ray treatment (a dose of 4 Gy). ( G ) Coimmunoprecipitation analysis of CRM1 interaction with YTHDF1 or S182A mutant. HEK293T cells transfected with the indicated plasmids were treated with or without x-ray irradiation (4 Gy). Cell lysates were subjected to immunoprecipitation using an anti–hemagglutinin (HA) antibody, followed by immunoblotting with both anti-HA and anti-FLAG antibodies.

Article Snippet: To achieve YTHDF1 ablation, single-guide RNA targeting human YTHDF1 was subcloned into the vector lentiCRISPR v2 (52961, Addgene) at the Bsm BI site.

Techniques: Phospho-proteomics, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Irradiation

Journal: Science Advances

Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response

doi: 10.1126/sciadv.ado7660

Figure Lengend Snippet: ( A ) Immunoblotting analysis of γH2AX levels in WT and YTHDF1-KO HEK293T cells treated with or without x-ray (4 Gy). ( B ) Immunofluorescence detection of γH2AX foci under the same conditions as (A). Scale bars, 10 μm. ( C ) Quantification of the number of γH2AX foci per cell in (B). ( D ) Neutral comet assays for detecting DNA damage under the same conditions as (A). Scale bars, 10 μm. ( E ) Quantification of the relative tail moments in (D) ( n > 50 cells). ( F ) Schematic of the reporter system for the analysis of HR-mediated DSB repair. ( G ) Schematic of the reporter system for the analysis of NHEJ-mediated DSB repair. ( H ) Quantification of the frequency of HR-mediated double-strand break repair in WT and YTHDF-KO cells. RFP, red fluorescent protein. ( I ) Quantification of the frequency of NHEJ-mediated double-strand break repair in WT and YTHDF-KO cells. ( J ) Cell proliferation assay of WT and YTHDF1-KO cells treated with or without x-ray (8 Gy; n = 5) using cell counting kit–8 (CCK-8). ( K ) Immunoblotting analysis of γH2AX level in the indicated cells treated with or without x-ray (4 Gy). YTHDF1-KO cells were reconstituted with either WT YTHDF1 or S182A mutant. ( L ) γH2AX foci number in WT cells, YTHDF-KO cells, or KO cells reconstituted with WT YTHDF1 or S182A mutant under x-ray treatment (4 Gy). ( M ) The relative tail moment in neutral comet assays under the same conditions as (L). ( N ) Cell proliferation assays of WT cells, YTHDF-KO cells, or KO cells reconstituted with WT YTHDF1 or S182A mutant under x-ray treatment (8 Gy) using CCK-8. All values represent the means ± SEM of three independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test (* P < 0.05, ** P < 0.01, and *** P < 0.001). ns, not significant.

Article Snippet: To achieve YTHDF1 ablation, single-guide RNA targeting human YTHDF1 was subcloned into the vector lentiCRISPR v2 (52961, Addgene) at the Bsm BI site.

Techniques: Western Blot, Immunofluorescence, Proliferation Assay, Cell Counting, CCK-8 Assay, Mutagenesis, Two Tailed Test

Journal: Science Advances

Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response

doi: 10.1126/sciadv.ado7660

Figure Lengend Snippet: ( A ) Schematic representation illustrating the strategy for the identification of YTHDF1-interacting proteins by LC/MS-MS. m / z , mass/charge ratio. ( B ) Functional enrichment analysis of 180 YTHDF1-interacting proteins based on the Database for Annotation, Visualization and Integrated Discovery analysis. ER, endoplasmic reticulum. ( C ) Overview of the assembly of the major spliceosome complex. ( D ) The spliceosome components that interact with YTHDF1. ( E ) Coimmunoprecipitation assay demonstrating the interaction between YTHDF1 and SF3B1, SF3B3, and SRSF2. HEK293T cells expressing FLAG-tagged YTHDF1 were treated with or without x-ray (4 Gy). Lysates were immunoprecipitated using IgG or anti-FLAG antibodies and subsequently analyzed by immunoblotting with the indicated antibodies. RNA digestion was performed using RNase A (5 μg/ml). ( F ) Impairment of the interaction between YTHDF1 and spliceosome components upon mutation of S182A. HEK293T cells expressing FLAG-YTHDF1 or S182A mutant were exposed to x-ray irradiation, followed by immunoprecipitation using an anti-FLAG antibody. ( G ) Colocalization of YTHDF1 with SRSF2 within nuclear speckles in response to x-ray treatment (4 Gy). Scale bars, 10 μm. a.u., arbitrary units.

Article Snippet: To achieve YTHDF1 ablation, single-guide RNA targeting human YTHDF1 was subcloned into the vector lentiCRISPR v2 (52961, Addgene) at the Bsm BI site.

Techniques: Liquid Chromatography with Mass Spectroscopy, Functional Assay, Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Irradiation

Journal: Science Advances

Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response

doi: 10.1126/sciadv.ado7660

Figure Lengend Snippet: ( A ) Venn diagram illustrating the overlap between m 6 A-containing transcripts (m 6 A targets) and YTHDF1-binding transcripts (CLIP targets). ( B ) Percentage representation of diverse RNAs bound by YTHDF1 based on CLIP-seq. Most binding clusters of YTHDF1 are located within mRNAs. ( C ) Distribution of YTHDF1-binding sites across four mRNA segments (intron, CDS, 5′UTR, and 3′UTR). ( D ) Boxplot depicting PSI changes following YTHDF1-KO for non-m 6 A exons, m 6 A-targeted exons, and YTHDF1-targeted exons. ( E ) Violin plot displaying exon expression changes following YTHDF1-KO for non-m 6 A exons, m 6 A-targeted exons, and YTHDF1-targeted exons. The upper and lower quartiles, as well as the median, are indicated for each group. ( F ) Functional enrichment analysis of transcripts in (C). ( G ) Integrative Genomics Viewer (IGV) tracks exhibiting the read coverage of BRCA1 and TP53BP1 genes from RNA-seq data of control (purple) and YTHDF1-KO (green) cells, along with CLIP-seq data (gray) and m 6 A data (blue). The RRACH sites located within m 6 A peaks are indicated. The normalized read number in the peak summit was indicated. ( H ) RT-PCR results and statistical analysis of exon inclusion level of BRCA1 and TP53BP1 in WT cells, YTHDF-KO cells, or KO cells reconstituted with WT YTHDF1 or S182A mutant under x-ray treatment (4 Gy). The ACTB gene was used as an internal control. ( I ) The mRNA level of BRCA1 , TP53BP1 , and RAD51 in WT cells, YTHDF-KO cells, or KO cells reconstituted with WT YTHDF1 or S182A mutant under x-ray treatment (4 Gy). ( J ) Immunoblotting analysis of WT cells, YTHDF-KO cells, or KO cells reconstituted with WT YTHDF1 or S182A mutant under x-ray treatment (4 Gy). Statistical significance was determined using the Mann-Whitney test (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: To achieve YTHDF1 ablation, single-guide RNA targeting human YTHDF1 was subcloned into the vector lentiCRISPR v2 (52961, Addgene) at the Bsm BI site.

Techniques: Binding Assay, Expressing, Functional Assay, RNA Sequencing, Control, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Western Blot, MANN-WHITNEY

Journal: Science Advances

Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response

doi: 10.1126/sciadv.ado7660

Figure Lengend Snippet: ( A ) Verification of SRSF2 knockdown (shSRSF2 #1 and shSRSF2 #2) efficiency in HEK293T cells. ( B ) Boxplot illustrating the PSI change in non-m 6 A exons, m 6 A-containing exons, and YTHDF1-targeted exons following SRSF2 knockdown (shSRSF2 #1). ( C ) Violin plot displaying the binding affinity of SRSF2 in YTHDF1-KO cells relative to the control for non-m 6 A exons, m 6 A-containing exons, and YTHDF1-targeted exons. The upper and lower quartiles, as well as the median, are indicated for each group. ( D ) Immunoblotting analysis of m 6 A-modified RNAs captured by FLAG-SRSF2 in the WT and YTHDF1-KO cells. WT or KO cells expressing FLAG-SRSF2 were cross-linked with UV, treated with micrococcal nuclease, and immunoprecipitated by FLAG antibody. The immunoprecipitates were subjected to immunoblotting analysis with m 6 A antibody. ( E and F ) IGV tracks exhibiting the read coverage of BRCA1 (E) and TP53BP1 (F) genes from RNA-seq data and SRSF2 RIP sequencing (RIP-seq) data. The m 6 A peaks are indicated. The normalized read number in the peak summit was indicated. ( G and H ) RT-PCR results and statistical analysis of exon inclusion level of BRCA1 (G) and TP53BP1 (H) in control and SRSF2-knockdown cells. The ACTB gene was used as an internal control. Statistical significance was determined using the Mann-Whitney test (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: To achieve YTHDF1 ablation, single-guide RNA targeting human YTHDF1 was subcloned into the vector lentiCRISPR v2 (52961, Addgene) at the Bsm BI site.

Techniques: Knockdown, Binding Assay, Control, Western Blot, Modification, Expressing, Immunoprecipitation, RNA Sequencing, Sequencing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Science Advances

Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response

doi: 10.1126/sciadv.ado7660

Figure Lengend Snippet: ( A ) Verification of YTHDF1 knockdown efficiency in HepG2 cells. ( B ) Cell proliferation assays conducted under normal conditions for control and YTHDF1-knockdown cells using CCK-8 ( n = 5). ( C ) Cell proliferation assays for control and YTHDF1-knockdown cells subjected to x-ray treatment (8 Gy; n = 5) using CCK-8. ( D ) Experimental design depicting the radiotherapy procedure in a mouse model. ( E ) Representative image of the tumors from control (shCTL) and YTHDF1-knockdown (shDF1) groups with or without x-ray irradiation (4 Gy). ( F ) Quantitative analysis of the tumor size and tumor volume in (E) ( n = 4). ( G ) Representative images of γH2AX staining in xenograft tumors subjected to x-ray treatment (4 Gy) and untreated controls with or without YTHDF1 depletion. Scale bar, 100 μm. ( H ) Quantitative analysis of γH2AX-positive cells in the samples shown in (G). ( I ) Representative images of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining in xenograft tumors subjected to x-ray treatment (4 Gy) and untreated controls with or without YTHDF1 depletion. Scale bar, 100 μm. ( J ) Quantitative analysis of the percentage of apoptotic cells (TUNEL-positive) in the samples presented in (I). All data are presented as means ± SEM from multiple independent experiments. Statistical significance was determined using an unpaired two-tailed Student’s t test (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: To achieve YTHDF1 ablation, single-guide RNA targeting human YTHDF1 was subcloned into the vector lentiCRISPR v2 (52961, Addgene) at the Bsm BI site.

Techniques: Knockdown, Control, CCK-8 Assay, Irradiation, Staining, End Labeling, TUNEL Assay, Two Tailed Test

Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: CVB3 infection induces cleavage of reader proteins YTHDF1-3 and increases CVB3 VP1 production and 2A transcription. HeLa cells were infected with CVB3 or sham-infected with PBS. Cell lysates were collected at 3 and 5 hpi for Western blot analysis to detect YTHDF2 ( A ), YTHDF1 ( B ), and YTHDF3 ( C ), as well as viral VP1. ( D ) HeLa cells were transfected with YTHDF2 siRNA (siY2) or control siRNA (siCtrl) and then infected with CVB3. ( G ) HeLa cells were transfected with a YTHDF2 plasmid (pY2) or an empty vector and then infected with CVB3. Western blot was conducted using the indicated antibodies ( D , G ), and protein levels were quantified using the ImageJ program ( E , H ). RT-qPCR was conducted using samples from ( D , G ) to measure the transcripts of the viral gene 2A ( F , I ). Data are presented as means ± SEM, n = 3, * p < 0.05, ** p < 0.01.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Infection, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m 6 A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.CAN-23-2081

Figure Lengend Snippet: Epi-Drug CRISPR dropout screens identify RUVBL1/2 as vulnerabilities of YTHDF1-expressing colorectal cancer cells. A, Composition of Epi-Drug sgRNA library and the workflow of CRISPR-Cas9 screens to identify YTHDF1-dependent vulnerabilities in colorectal cancer cells. B, Principal component analysis (PCA) of sgRNA abundances in each group at the end point of CRISPR-Cas9 screening. C, Left, top depleted genes in YTHDF1-overexpressing DLD1 cells vs. control vector (log 2 (fold change) < −0.5; log 10 ( P value < −1). Middle, top enriched genes in shYTHDF1 cells vs. shControl (log 2 (fold change) > 0.5; log 10 ( P value < −1). Right, overlapping of outlier genes identified the common candidates preferentially essential in a YTHDF1-dependent fashion. D and E, RUVBL1/2 mRNA expression in colorectal cancer cells compared with adjacent normal tissues in Hong Kong ( D ) and TCGA ( E ) colorectal cancer cohorts. In Hong Kong cohort, mRNA expression was normalized to β-actin. F, RUVBL1/2 and YTHDF1 proteins are overexpressed in colorectal cancer cells compared with paired adjacent normal tissues. G, Left, representative images of YTHDF1, RUVBL1, and RUVBL2 staining in colorectal cancer tissue microarrays ( N = 184). Right, Pearson correlation analysis of YTHDF1, RUVBL1, and RUVBL2 protein expression. H, Left, Kaplan–Meier curve analysis of RUVBL1 protein expression and patient survival in colorectal cancer in tissue microarray cohort ( N = 184). Right, multivariate Cox regression analysis. RUVBL1-low, IHC score 1; RUVBL1-high, IHC score 2 to 3. I, Left, Kaplan–Meier curve analysis of RUVBL2 protein expression and colorectal cancer patient survival. Right, multivariate Cox regression analysis. RUVBL2-low, IHC score 1 to 2; RUVBL2-high, IHC score 3. Paired t test ( D and E ; left), Student t -test ( E ; right), Pearson χ 2 test ( G ), or log rank test ( H and I ).

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: CRISPR, Expressing, Control, Plasmid Preparation, Staining, Microarray

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m 6 A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.CAN-23-2081

Figure Lengend Snippet: RUVBL1/2 knockout abolishes oncogenic function of YTHDF1 in vitro and in vivo . A–D, Effect of RUVBL1/2 knockout on vector- and YTHDF1-overexpressing DLD1 and HCT116 cell proliferation ( N = 10; A ), colony formation ( N = 3, 7–14 days; B ), apoptosis ( N = 3; C ), and G 1 -S cell cycle transition ( N = 3; D ). E, Western blot of cell cycle and apoptosis markers. F, Representative brightfield images of primary colorectal cancer tumor-derived organoids expressing vector or YTHDF1, with or without RUVBL1/2 knockout. G, Effect of RUVBL1/2 knockout on vector- and YTHDF1-overexpressing DLD1 and HCT116 xenografts in nude mice. RUVBL1/2 abrogated differential growth between vector- and YTHDF1-overexpresing xenografts (DLD1, N = 5; HCT116, N = 8). Two-way ANOVA ( A ) and one-way ANOVA ( B–D and G ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: Knock-Out, In Vitro, In Vivo, Plasmid Preparation, Western Blot, Derivative Assay, Expressing

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m 6 A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.CAN-23-2081

Figure Lengend Snippet: YTHDF1 directly targets m 6 A-modified RUVBL1/2 mRNA methylation and promotes their protein expression in vitro and in vivo . A, UCSC snapshots of m 6 A-seq reads of RUVBL1/2 mRNA in DLD1 cells. The normalized read densities are shown for m 6 A (orange) and input (blue). B, Methylated RIP-qPCR analysis of m 6 A-modified RUVBL1/2 mRNA in DLD1 and HCT116 cells. C, RIP-qPCR with anti-YTHDF1 antibody showed binding of YTHDF1 to RUVBL1/2 mRNA, whereas mutant YTHDF1 (K395A, Y397A) had attenuated binding. D and E , Effect of YTHDF1 overexpression ( D ) or knockdown ( E ) on RUVBL1/2 mRNA and protein expression in DLD1 and HCT116 cells. F, Effect of YTHDF1 overexpression on RUVBL1/2 protein expression in primary colorectal cancer organoids PDO828 and PDO74. G, Expression of YTHDF1 and RUVBL1/2 in intestinal-specific Ythdf1 knockin mice (Ythdf1 lsl Cdx2-Cre ERT2 ) as compared with wildtype mice. Student t test ( B–D ) and one-way ANOVA ( E ). ****, P < 0.0001.

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: Modification, Methylation, Expressing, In Vitro, In Vivo, Binding Assay, Mutagenesis, Over Expression, Knockdown, Knock-In

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m 6 A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.CAN-23-2081

Figure Lengend Snippet: YTHDF1 promotes translation efficiency of RUVBL1/2, which in turn interact with YTHDF1 and translational initiation factors. A, RNC-qPCR analysis of ribosome-associated RUVBL1/2 mRNA in vector- and YTHDF1-overexpressing DLD1 and HCT116 cells. B, Enrichment of RUVBL1/2 mRNA in < 40S, 40S, 60S, 80S, and polysomes from HCT116 cells with or without YTHDF1 overexpression. C and D, Colorectal cancer cells overexpressing wildtype YTHDF1 or mutant YTHDF1 were transfected with pmirGLO-RUVBL1 ( C ) or pmirGLO-RUVBL2 ( D ) containing respective 3′UTR sequences, followed by luciferase assays. E, pmirGLO-RUVBL1/2-mutant reporters with mutated m 6 A sites (RRACH to TTTCT) in the 3′UTR region demonstrated decreased luciferase activity. F, RUVBL1/2 coimmunoprecipitation and mass spectrometry for identification of common interacting proteins. G, Pathway enrichment analysis [gene ontology (GO), GSEA-KEGG] of interacting partners of RUVBL1/2. H, Coimmunoprecipitation by anti-YTHDF1 verified binding of YTHDF1 to RUVBL1/2. I, Coimmunoprecipitation using recombinant YTHDF1 and RUVBL1/2 confirmed direct protein–protein interplay between YTHDF1 and RUVBL1/2. J, Colocalization of RUVBL1/2 and YTHDF1 in HCT116 cells was determined by immunofluorescence staining. Student t test ( A , B , and E ) and one-way ANOVA ( C and D ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: Plasmid Preparation, Over Expression, Mutagenesis, Transfection, Luciferase, Activity Assay, Mass Spectrometry, Binding Assay, Recombinant, Immunofluorescence, Staining

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m 6 A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.CAN-23-2081

Figure Lengend Snippet: RUVBL1/2 knockout abrogated YTHDF1-induced translation initiation and oncogenic signaling. A, Left, polysome profiling of HCT116 cells with overexpression of YTHDF1 with or without knockout of RUVBL1/2. Right, Western blot of ribosomal fractions (<40S, 40S, 60S, 80S and polysomes). B, Stress granules (SG) were determined by immunofluorescence staining of TIA1-related protein (TIAR). C, HPG protein incorporation assay for the detection of nascent protein synthesis by immunofluorescence staining. D, Puromycin incorporation assay of protein synthesis. E, Ribo-seq of YTHDF1-overexpressing HCT116 cells with or without RUVBL1/2 knockout, following GSEA-KEGG pathway enrichment analysis. F, Effect of RUVBL1/2 knockout on the translation efficiency of MAP3K2, MAP3K7, MAPK8IP1, and ETS2 in HCT116 cells with YTHDF1 overexpression. G, Western blot of MAPK and PI3K-Akt signaling markers. One-way ANOVA ( B and C ). ***, P < 0.001; ****, P < 0.0001.

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: Knock-Out, Over Expression, Western Blot, Immunofluorescence, Staining

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m 6 A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.CAN-23-2081

Figure Lengend Snippet: Pharmacological RUVBL1/2 inhibitor inhibits the growth of YTHDF1-overexpressing colorectal cancer cells. A, Structure of a RUVBL1/2 complex inhibitor, CB6644. B, Forty-eight hours-IC 50 values indicated that CB6644 preferentially inhibited the growth of DLD1 and HCT116 cells with YTHDF1 overexpression. C, CB6644 preferentially impaired colony formation capacity in YTHDF1-overexpressing DLD1 and HCT116 cells (7–14 days). D, CB6644 (0.5 µmol/L for DLD1; 0.1 µmol/L for HCT116, 24 hours) abrogated suppressive effect of YTDHF1 overexpression on apoptosis. Puromycin (0.5 µg/mL, 24 hours) was used as positive control. E, Treatment of DLD1 cells with CB6644 (0.5 µmol/L, 36 hours), followed by coimmunoprecipitation to analyze their interactions with YTHDF1. F, Interaction between YTHDF1 and EIF3K or EIF4A after treatment with CB6644 in DLD1 cells (0.5 µmol/L, 36 hours). G, Effect of CB6644 on protein translation in DLD1 cells, as assessed by puromycin incorporation assay (0.5 µmol/L, 6 hours). H, DLD1 cells expressing sgRUVBL1 or sgRUVBL2 were overexpressed with wildtype or ATPase-dead mutant RUVBL1 or RUVBL2, respectively. Coimmunoprecipitation was performed with anti-YTHDF1 to determine its interaction with RUVBL1/2, EIF3K, and EIF4A. I, Effect of ATPase-dead mutant RUVBL1 or RUVBL2 on protein translation in DLD1 cells compared with wildtype counterparts. One-way ANOVA ( E and F ). ****, P < 0.0001.

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: Over Expression, Positive Control, Expressing, Mutagenesis

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m 6 A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.CAN-23-2081

Figure Lengend Snippet: In vivo efficacy of RUVBL1/2 inhibitors or vesicle-like nanoparticle-encapsulated siRUVBL1/2. A, DLD1 vector- or YTHDF1-overexpressing xenografts were treated with CB6644 (25 mg/kg, i.t.; arrows). B, HCT116 vector- or YTHDF1-overexpressing xenografts were treated with CB6644 (25 mg/kg, i.t.; arrows). C, Ki67 staining of DLD1 xenografts treated with CB6644. D, Structure of si-RUVBL1/2 encapsulated by VNPs. E, VNP-siRUVBL1/2 knockdown efficiency was confirmed in HCT116 cells in vitro . F, Effect of VNP-siRUVBL1/2 (2 mg/kg, i.t.; arrows) on DLD1 xenografts with or without YTHDF1 overexpression. G, Effect of VNP-siRUVBL1/2 (2 mg/kg, i.t.; arrows) on HCT116 xenografts with or without YTHDF1 overexpression. H, Ki67 staining of DLD1 xenografts treated with VNP-siRUVBL1/2. I, Schematic diagram showing the mechanism of RUVBL1/2 blockade in YTHDF1-expressing cells. RUVBL1/2 forms a complex with YTHDF1 and associated translation initiation factors, which is essential for YTHDF1-induced protein translation and oncogenic signaling. RUVBL1/2 themselves are targets of YTHDF1, forming a feedforward circuitry that boosts translation in colorectal cancer. RUVBL1/2 inhibition arrested translation by YTHDF1 and abrogated YTHDF1-induced oncogenic signaling and tumorigenesis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ( D and I, Created with BioRender.com .)

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: In Vivo, Plasmid Preparation, Staining, Knockdown, In Vitro, Over Expression, Expressing, Inhibition

Journal: Experimental and Therapeutic Medicine

Article Title: YTHDF1 regulates immune cell infiltration in gastric cancer via interaction with p53

doi: 10.3892/etm.2024.12543

Figure Lengend Snippet: YTHDF1 expression in GC is elevated and associated with pathological features. (A) Genetic alterations in the YTH domain family in GC were analyzed using the cBioPortal database. YTHDF1 was overexpressed in GC tissue compared with adjacent normal tissue analyzed with the (B) Gene expression profiling interactive analysis and (C) University of Alabama at Birmingham Cancer data analysis portal databases. (D) YTHDF1 expression was associated with tumor grade in GC analyzed by UALCAN database. (E) YTHDF1 expression was associated with tumor grade in GC analyzed by TISIDB database (F) YTHDF1 expression was associated with microsatellite status in GC. (G) Associations between YTHDF1 and molecular subtypes in GC were analyzed using the Tumor-immune system interactions and drug bank database. (H) YTHDF1 expression in GC cell lines was analyzed using reverse transcription-quantitative PCR. (I) Differences in YTHDF1 expression between GC and paired normal tissue was analyzed using western blotting. * P<0.05; *** P<0.001. YTHDF1, YTH N 6 -methyladenosine RNA binding protein 1; GC, gastric cancer; STAD, stomach adenocarcinoma; T, tumor; N, normal; TPM, transcript per million; CPM, counts per million reads; CPTAC, Clinical Proteomic Tumor Analysis Consortium; PCR, polymerase chain reaction.

Article Snippet: Subsequently, the sections were incubated with rabbit anti-human polyclonal YTHDF1 antibodies (1:100; cat. no. 17479-1-AP; Proteintech Group, Inc.) in a humidified box overnight at 4 ̊C.

Techniques: Expressing, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, RNA Binding Assay, Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: YTHDF1 regulates immune cell infiltration in gastric cancer via interaction with p53

doi: 10.3892/etm.2024.12543

Figure Lengend Snippet: Prognostic value of YTHDF1 in different types of human cancers. Kaplan-Meier analysis suggested that upregulation of YTHDF1 indicates poor prognosis in patients with (A) gastric, (B) liver, (C) thyroid, (D) breast, (E) ovarian cancers and (F) endometrial carcinoma. YTHDF1, YTH N 6 -methyladenosine RNA binding protein 1; HR, hazard ratio.

Article Snippet: Subsequently, the sections were incubated with rabbit anti-human polyclonal YTHDF1 antibodies (1:100; cat. no. 17479-1-AP; Proteintech Group, Inc.) in a humidified box overnight at 4 ̊C.

Techniques: RNA Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: YTHDF1 regulates immune cell infiltration in gastric cancer via interaction with p53

doi: 10.3892/etm.2024.12543

Figure Lengend Snippet: Correlation between YTHDF1 expression and immune infiltration level. (A) Spearman's correlations between YTHDF1 expression and TILs across several human cancer types. Correlations between YTHDF1 expression and specific immune cell types in gastric cancer, including (B) B cells, (C) macrophages, (D) DCs, (E) monocytes, (F) CD8+ T cells, (G) neutrophils, (H) Tregs, (I) CD4+ T cells, (J) Treg cells and (K) MDSC cells, were analyzed using the Tumor-Immune System Interactions and Drug Bank database. (L) Correlation between YTHDF1 expression and immune infiltration levels in gastric cancer was analyzed using the TIMER database. (M) Comparison of immune infiltration levels between gastric cancer with or without YTHDF1 copy number alterations was performed using the SCNA module of the TIMER database. * P<0.05; ** P<0.01. YTHDF1, YTH N 6 -methyladenosine RNA binding protein 1; TILs, tumor-infiltrating lymphocytes; DCs, dendritic cells; Tregs, regulatory T cells; MDSCs, myeloid-derived suppressor cells; TIMER, Tumor Immune Estimation Resource; STAD, stomach adenocarcinoma; Act_CD8, activated CD8 T cells; Tcm_CD8, central memory CD8 T cells; Tem_CD8, effector memory CD8 T cells; Tcm_CD4, central memory CD4 T cells; Tem_CD4, effector memory CD4 T cells; Act_B, activated B cells; Imm_B, immune B cells; TPM, transcripts per million.

Article Snippet: Subsequently, the sections were incubated with rabbit anti-human polyclonal YTHDF1 antibodies (1:100; cat. no. 17479-1-AP; Proteintech Group, Inc.) in a humidified box overnight at 4 ̊C.

Techniques: Expressing, Comparison, RNA Binding Assay, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: YTHDF1 regulates immune cell infiltration in gastric cancer via interaction with p53

doi: 10.3892/etm.2024.12543

Figure Lengend Snippet: Lymphocyte subsets enrichment in GC specimens. (A) Representative immunohistochemistry images of different YTHDF1 expression levels in GC specimens (x100 magnification, stained with liquid DAB). Difference in the lymphocyte subsets infiltration level in GC specimens with high and low levels of YTHDF1 expression: (B) Total lymphocyte, (C) total T cell, (D) CD4 + T cell, (E) CD8 + T cell, (F) NK cell and (G) B cell. ** P<0.01; *** P<0.001. GC, gastric cancer; YTHDF1, YTH N 6 -methyladenosine RNA binding protein 1; NK, natural killer; ns, not significant.

Article Snippet: Subsequently, the sections were incubated with rabbit anti-human polyclonal YTHDF1 antibodies (1:100; cat. no. 17479-1-AP; Proteintech Group, Inc.) in a humidified box overnight at 4 ̊C.

Techniques: Immunohistochemistry, Expressing, Staining, RNA Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: YTHDF1 regulates immune cell infiltration in gastric cancer via interaction with p53

doi: 10.3892/etm.2024.12543

Figure Lengend Snippet: Correlation between YTHDF1 and markers of immune cells in gastric cancer. Correlation between YTHDF1 and (A) tumor associate macrophage-related genes and markers and (B) DC markers were analyzed using the Gene module of the Tumor Immune Estimation Resource database. Correlation between YTHDF1 and Treg markers, including (C) Foxp3, (D) TGFB1 and (E) CCR8 were analyzed using the GEPIA database. Correlation between YTHDF1 and T cell exhaustion markers, namely (F) PDCD1, (G) CTLA4 and (H) HAVCR2 (T cell immunoglobulin and mucin-domain-containing-3) were analyzed using the GEPIA database. YTHDF1, YTH N 6 -methyladenosine RNA binding protein 1; CSF1, colony-stimulating factor 1; STAT, signal transducer and activator of transcription; TPM, transcripts per million; DC, dendritic cell; Foxp3, forkhead box P3; TGFB1, transforming growth factor-β1; CCR8, C-C motif chemokine receptor; PDCD1, programmed cell death 1; CTLA-4, T-lymphocyte-associated protein 4; HAVCR2, hepatitis A virus cellular receptor 2; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: Subsequently, the sections were incubated with rabbit anti-human polyclonal YTHDF1 antibodies (1:100; cat. no. 17479-1-AP; Proteintech Group, Inc.) in a humidified box overnight at 4 ̊C.

Techniques: RNA Binding Assay, Virus, Gene Expression

Journal: Experimental and Therapeutic Medicine

Article Title: YTHDF1 regulates immune cell infiltration in gastric cancer via interaction with p53

doi: 10.3892/etm.2024.12543

Figure Lengend Snippet: Interactions between YTHDF1 and p53. Correlation between YTHDF1 and the gastric-related genes (A) TP53, (B) ERBB2 (HER-2) and (C) KRAS were analyzed using the Gene Expression Profiling Interactive Analysis database. (D) YTHDF1 expression was elevated in TP53 mutant gastric cancer compared with TP53 nonmutant gastric cancer, as analyzed using the University of Alabama at Birmingham Cancer data analysis portal database. (E) TP53 mutation was associated with decreased immune cell infiltration levels in gastric cancer, as analyzed by the Tumor Immune Estimation Resource database. (F) p53 interaction with YTHDF1 was analyzed using the GeneMANIA database. (G) p53 interaction with YTHDF1 was analyzed using the HitPredict database. (H) Interactions between YTHDF1 and p53 were assessed using immunoprecipitation assays in 293T cells transfected with plasmids, followed by co-immunoprecipitation assays using anti-Myc antibodies. (I) Interactions between YTHDF1 and p53 were assessed using immunoprecipitation assays in 293T cells transfected with plasmids, followed by co-immunoprecipitation assays using anti-p53 antibodies. * P<0.05; ** P<0.01; *** P<0.001. YTHDF1, YTH N 6 -methyladenosine RNA binding protein 1; TP53, tumor protein p53; ERBB2, erb-b2 receptor tyrosine kinase 2; HER-2, human epidermal growth factor receptor-2; KRAS, KRAS proto-oncogene; TPM, transcripts per million; TCGA, The Cancer Genome Atlas; WT, wild type; STAD, stomach adenocarcinoma.

Article Snippet: Subsequently, the sections were incubated with rabbit anti-human polyclonal YTHDF1 antibodies (1:100; cat. no. 17479-1-AP; Proteintech Group, Inc.) in a humidified box overnight at 4 ̊C.

Techniques: Gene Expression, Expressing, Mutagenesis, Immunoprecipitation, Transfection, RNA Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: YTHDF1 regulates immune cell infiltration in gastric cancer via interaction with p53

doi: 10.3892/etm.2024.12543

Figure Lengend Snippet: Enrichment analysis of YTHDF1 functional networks in gastric cancer using the LinkedOmics database. (A) Volcano plot illustrating upregulated (red) and downregulated (green) genes. Heatmap showing the top 50 genes (B) positively associated with YTHDF1 and (C) negatively associated with YTHDF1. (D) KEGG analysis, (E) cellular component analysis, (F) biological process analysis and (G) molecular function analysis of YTHDF1 was performed using the Gene Set Enrichment Analysis tool. YTHDF1, YTH N 6 -methyladenosine RNA binding protein 1; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Subsequently, the sections were incubated with rabbit anti-human polyclonal YTHDF1 antibodies (1:100; cat. no. 17479-1-AP; Proteintech Group, Inc.) in a humidified box overnight at 4 ̊C.

Techniques: Functional Assay, RNA Binding Assay

Journal: Cell Death Discovery

Article Title: METTL3-mediated deficiency of lncRNA HAR1A drives non-small cell lung cancer growth and metastasis by promoting ANXA2 stabilization

doi: 10.1038/s41420-024-01965-w

Figure Lengend Snippet: A , B RT-qPCR was performed to investigate the effects of overexpression and knockdown of METTL3 on the HAR1A levels in A549 and H1299 cells. C After inhibiting RNA synthesis with actinomycin D, HAR1A degraded faster in NSCLC cells with METTL3 overexpression than in control vector cells at different times. D m 6 A RIP coupled with RT-qPCR showed that HAR1A was subjected to m 6 A modification, and significantly more m 6 A-modified HAR1A RNAs were enriched in A549 and H1299 than in HBE cells. E Cells were transduced with siRNAs targeting YTHDF1, YTHDF2, YTHDF3, and scramble controls. siYTHDF2 treatment reduced HAR1A levels as shown by RT-qPCR. F In cells where RNA synthesis was blocked with actinomycin D, YTHDF2 siRNA slowed down the degradation of HAR1A . G RIP assay, followed by RT-qPCR, revealed the precipitation of YTHDF2 with HAR1A . H Schematic diagram of molecular mechanisms. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Short interference RNAs (siRNAs) against human METTL3 (siMETTL3), YTHDF1 (siYTHDF1), YTHDF2 (siYTHDF2), YTHDF3 (siYTHDF3) and matched negative controls (siNC) were provided by Genechem (Shanghai, China).

Techniques: Quantitative RT-PCR, Over Expression, Knockdown, Control, Plasmid Preparation, Modification, Transduction