Journal: International Journal of Molecular Sciences

Article Title: Ebselen Suppresses Breast Cancer Tumorigenesis by Inhibiting YTHDF1-Mediated c-Fos Expression

doi: 10.3390/ijms26199416

Figure Lengend Snippet: YTHDF1 overexpression in breast cancer represents a viable therapeutic target for ebselen. ( a ) Comparison of RNA expression levels of the YTHDF gene family in breast cancer tissues and matched adjacent normal tissues, based on RNA-seq data from The Cancer Genome Atlas (TCGA). ( b ) Kaplan–Meier plot showing the association between YTHDF1 expression and progression-free survival in the TCGA breast cancer cohort. Patients with breast cancer ( n = 100 per group) were stratified into high and low YTHDF1 expression groups (data for panels ( a , b ) were retrieved from https://portal.gdc.cancer.gov/projects/TCGA-BRCA (accessed on 6 July 2025)). ( c ) Structural model showing ebselen binding to the YTH domain of YTHDF1, specifically near the m 6 A recognition pocket, forming a covalent bond with the Cys412 residue (PDB ID: 7PCU; structure retrieved from https://www.rcsb.org/ (accessed on 8 July 2025)). ( d ) Ebselen induces concentration-dependent cytotoxicity. In MCF7 cells. Cells were treated with the indicated concentrations of ebselen for 24 or 48 h, and cell viability was measured using the MTT assay.

Article Snippet: Antibodies against c-Fos (Clone 9F6; 2250; WB 1:1000), METTL3 (Clone E3F2A; 86132; WB 1:1000), Beclin-1 (3738; WB 1:1000), and YTHDF1 (86463S; WB 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Over Expression, Comparison, RNA Expression, RNA Sequencing, Expressing, Binding Assay, Residue, Concentration Assay, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Ebselen Suppresses Breast Cancer Tumorigenesis by Inhibiting YTHDF1-Mediated c-Fos Expression

doi: 10.3390/ijms26199416

Figure Lengend Snippet: Ebselen regulates YTHDF1-mediated c-Fos expression in MCF7 cells. ( a ) Volcano plot showing mRNAs upregulated by epidermal growth factor (EGF) treatment in a published RNA-seq dataset, with significantly upregulated transcripts highlighted in red. ( b , c ) Upregulation of FOS gene expression by EGF. MCF7 cells were cultured in serum-free medium for 24 h and treated with EGF for 1 h. Expression of FOS family genes was analyzed using real-time ( b ) or end-point ( c ) PCR with gene-specific primers. Data are presented as mean ± SD; n = 3. One-way analysis of variance (ANOVA), *** p < 0.001. ( d ) Ebselen treatment decreases FOS mRNA and protein levels in a concentration-dependent manner. MCF7 cells were treated with varying concentrations of ebselen in serum-free media for 24 h, followed by EGF stimulation. mRNA ( upper panel ) and protein ( lower panel ) expression was analyzed using end-point PCR and western blotting (WB) for the indicated target genes and proteins, respectively. ( e ) Knockout of YTHDF1 reduces FOS mRNA and c-Fos protein levels. MCF7 cells transfected with sgCtrl or sgYTHDF1 were serum-starved and treated with EGF. mRNA ( upper panel ) and protein ( lower panel ) expression was analyzed for the indicated target genes and proteins, respectively. ( f ) RNA immunoprecipitation (RIP) analysis using anti-YTHDF1 antibody confirmed the interaction between FOS mRNA and YTHDF1 protein. MCF7 cells were serum-starved and treated with varying concentrations of ebselen for 24 h, followed by EGF stimulation. Cell lysates were then subjected to RIP, and the associated RNA was analyzed via PCR. ( g , h ) Methyltransferase-like 3 (METTL3) regulates EGF-induced FOS expression. MCF7 cells transfected with sgCtrl or sgMETTL3 were serum-starved for 24 h ( g ). or wild-type cells were pre-treated with STM2457 (a METTL3 inhibitor) in serum-free medium ( h ). In both cases, cells were then stimulated with EGF for 1 h, followed by end-point PCR ( upper panel ) and WB ( lower panel ) analysis for the indicated target genes and proteins, respectively.

Article Snippet: Antibodies against c-Fos (Clone 9F6; 2250; WB 1:1000), METTL3 (Clone E3F2A; 86132; WB 1:1000), Beclin-1 (3738; WB 1:1000), and YTHDF1 (86463S; WB 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, RNA Sequencing, Gene Expression, Cell Culture, Concentration Assay, Western Blot, Knock-Out, Transfection, RNA Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Ebselen Suppresses Breast Cancer Tumorigenesis by Inhibiting YTHDF1-Mediated c-Fos Expression

doi: 10.3390/ijms26199416

Figure Lengend Snippet: Ebselen reduces FOS mRNA stability. ( a , b ) Effect of ebselen on FOS promoter activity. Schematic representation of the pGL5-FOS reporter construct containing the serum response element (indicated as SRE) consensus sequence in the promoter region of the luciferase reporter gene ( a ). MCF7 cells were transfected with the pGL5-FOS-luc reporter plasmid, followed by ebselen treatment and EGF stimulation ( b ). Firefly luciferase activity was then measured to assess FOS promoter activity. Data are presented as mean ± SD; n = 3. One-way ANOVA, ### p < 0.001. ( c , d ) Knockout of YTHDF1 reduces FOS mRNA stability. MCF7 cells transfected with sgControl or sgYTHDF1 were treated with actinomycin D (indicated as ActD) for the indicated time points. FOS mRNA expression was analyzed using end-point PCR ( c ). Band intensities were quantified via densitometry and normalized to that of GAPDH ( d ). Data are presented as mean ± SD; n = 3. Student’s t -test, * p < 0.05, *** p < 0.001 (sgControl versus sgYTHDF1 at corresponding time points), # p < 0.05 (sgControl, 0 min versus 60 min) ( d ). ( e , f ) Ebselen treatment reduces FOS mRNA stability. MCF7 cells were pretreated with ebselen (50 µM) for 24 h, followed by actinomycin treatment at the indicated time points. FOS mRNA expression was analyzed using end-point PCR ( e ), and band intensities were quantified via densitometry and normalized to that of GAPDH ( f ). Data are presented as mean ± SD; n = 3. Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001 (DMSO versus ebselen at corresponding time points), ## p < 0.01 (DMSO, 0 min versus 60 min).

Article Snippet: Antibodies against c-Fos (Clone 9F6; 2250; WB 1:1000), METTL3 (Clone E3F2A; 86132; WB 1:1000), Beclin-1 (3738; WB 1:1000), and YTHDF1 (86463S; WB 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Activity Assay, Construct, Sequencing, Luciferase, Transfection, Plasmid Preparation, Knock-Out, Expressing

Journal: Cell death & disease

Article Title: CircMIB2 therapy can effectively treat pathogenic infection by encoding a novel protein.

doi: 10.1038/s41419-023-06105-3

Figure Lengend Snippet: Fig. 6 N6-methyladenosine modification mediates circMIB2 translation proteins. A Myc-TRAF6 and Flag-circMIB2-P were co-transfected into MSpC cells with m6A modification-related genes, respectively, and then the protein levels of Myc-TRAF6 and Flag-circMIB2 were detected. B–D Myc-TRAF6 and Flag-circMIB2-P were co-transfected into MSpC cells with METTL14 or METTL16 respectively (B), Myc-TRAF6 and Flag- circMIB2-P were co-transfected into MSpC cells with YTHDF1 and YTHDF3 respectively (C), Myc-TRAF6 and Flag-circMIB2-P were co- transfected into MSpC cells with ALKBH5 (D), and then the protein levels of Myc-TRAF6 and Flag-circMIB2 were detected. E The level of circMIB2 upon YTHDF1 or YTHDF3 overexpression was examined by RIP-qPCR. F The m6A level alteration of circMIB2 upon METTL14, METTL16, or ALKBH5 overexpression were examined by MeRIP-qPCR. G Relative RNA levels of circMIB2 in MSpC cells after transfected with pcDNA3.1, METTL14, METTL16, YTHDF1, YTHDF3, and ALKBH5, respectively. H The protein level of YTHDF1 or YTHDF3 was examined by RNA pulldown. All data represented the mean ± SE from three independent triplicated experiments. *p < 0.05; **p < 0.01.

Article Snippet: The MS2RNA pulldown assay was conducted in MSpC cells transfected with YTHDF1, YTHDF3, pMS2-GFP (Addgene) plasmids and MS2-circMIB2, MS2-circMIB2m6A-mut circRNA, and the specific steps were performed as described [30].

Techniques: Transfection, Over Expression

Journal: Zoological research

Article Title: Single-cell sequencing analysis reveals the essential role of the m 6 A reader YTHDF1 in retinal visual function by regulating TULP1 and DHX38 translation.

doi: 10.24272/j.issn.2095-8137.2024.399

Figure Lengend Snippet: Figure 1 YTHDF1 deficiency impairs visual function and leads to progressive retinal degeneration

Article Snippet: Conventional Ythdf1 KO mice were generated using the CRISPR/Cas9 system by Suzhou Cyagen Biosciences Inc. (Suzhou, China).

Techniques:

Journal: Zoological research

Article Title: Single-cell sequencing analysis reveals the essential role of the m 6 A reader YTHDF1 in retinal visual function by regulating TULP1 and DHX38 translation.

doi: 10.24272/j.issn.2095-8137.2024.399

Figure Lengend Snippet: Figure 3 Loss of Ythdf1 impairs visual function and leads to progressive degeneration of rod cells

Article Snippet: Conventional Ythdf1 KO mice were generated using the CRISPR/Cas9 system by Suzhou Cyagen Biosciences Inc. (Suzhou, China).

Techniques:

Journal: Zoological research

Article Title: Single-cell sequencing analysis reveals the essential role of the m 6 A reader YTHDF1 in retinal visual function by regulating TULP1 and DHX38 translation.

doi: 10.24272/j.issn.2095-8137.2024.399

Figure Lengend Snippet: Figure 5 Transcriptome-wide identification of YTHDF1 targets in retinal rod cells

Article Snippet: Conventional Ythdf1 KO mice were generated using the CRISPR/Cas9 system by Suzhou Cyagen Biosciences Inc. (Suzhou, China).

Techniques:

Journal: Zoological research

Article Title: Single-cell sequencing analysis reveals the essential role of the m 6 A reader YTHDF1 in retinal visual function by regulating TULP1 and DHX38 translation.

doi: 10.24272/j.issn.2095-8137.2024.399

Figure Lengend Snippet: Figure 6 YTHDF1 promotes translation of m6A-methylated Tulp1 and Dhx38 in retinas

Article Snippet: Conventional Ythdf1 KO mice were generated using the CRISPR/Cas9 system by Suzhou Cyagen Biosciences Inc. (Suzhou, China).

Techniques: Methylation

Journal: Genome biology

Article Title: ADAR1 is a new target of METTL3 and plays a pro-oncogenic role in glioblastoma by an editing-independent mechanism.

doi: 10.1186/s13059-021-02271-9

Figure Lengend Snippet: Fig. 3 YTHDF1 binds m6A on ADAR1 mRNA boosting its translation without affecting decay. a Box plot showing YTHDF1 mRNA levels in GBM (red box) versus normal brain (blue box) (GEPIA 2, * p ≤0.05). b qRT- PCR of YTHDF1 and ADAR1 in siYTHDF1 U87MG cells (24–48 h post transfection-pt). On the right, western blotting analysis (48 h pt) of ADAR1 in siYTHDF1 U87MG cells is shown. GAPDH was used as control. c Relative enrichment of ADAR1 mRNA in YTHDF1-RIP (using two different antibodies Ab1 and Ab2) over IgG in U87MG cells. HPRT expression was used as negative control (n = 3) Values are represented as means ± SD, * p ≤0.05. d Ribosomal immunoprecipitation was performed in siYTHDF1 and siscr U87MG cells transfected with an RPL22-FLAG construct. Left, is shown a control western blotting analysis, while on the right, a qRT-PCR is shown. HPRT was used as control (n = 3). Values are represented as means ± SD, ** p ≤0.01. e ADAR1 mRNA stability in control and siYTHDF1 U87MG cells was determined by qRT-PCR after actinomycin D (5 μg/ml) treatment in the time indicated. Values are represented as means ± SD. On the right, the qRT-PCR of YTHDF1 silencing. Values are represented as means ± SD, *** p ≤0.001, n = 2. f Western blotting analysis of siYTHDF1 and control U87MG cells treated with or without MG132 at different concentrations (indicated in the figure); ADAR2 and ubiquitin Ab (Ubi) were used as controls. On the right, siYTHDF1 and sictrl U87MG cells were treated with or without 1.25 μM MG132 for 24 h. GAPDH was used as loading control. Values are represented as means ± SD, * p ≤0.05

Article Snippet: The antibodies used were as follows: ADAR1 (Santa Cruz Biotechnology), ADAR1 (Bethyl), CDK2 (Santa Cruz Biotechnology), YTHDF1 (Abcam), METTL3 (Abcam), METTL14 (Bethyl), cyclinE (Santa Cruz Biotechnology), p57 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), CDC14B (LifeSpan), ADAR2 (Santa Cruz Biotechnology), Ubiquitin (Thermo Fisher), β-actin (Santa Cruz Biotechnology), GAPDH (Cell Signaling), and the anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Control, Expressing, Negative Control, Immunoprecipitation, Construct, Ubiquitin Proteomics

Journal: Genome biology

Article Title: ADAR1 is a new target of METTL3 and plays a pro-oncogenic role in glioblastoma by an editing-independent mechanism.

doi: 10.1186/s13059-021-02271-9

Figure Lengend Snippet: Fig. 7 Targeting ADAR1 in growing tumor mass blocks glioblastoma progression. a Quantitative analysis of tumor size (tumor volume) of U87MG cells subcutaneously injected into the flank of NOD-SCID mice (n = 16 mice). Tumors generated by control (n = 8) or shADAR1 (n = 8) inducible U87MG cells were treated with DOXY (in drinking water). Tissues were collected and analyzed at the end of treatment (60 days p.i.). *p ≤0.05. Right, a representative picture of tumors. Representative sections and relative quantification of Ki67 (b), ADAR1 (c), and CDK2 (d) staining are shown. e qRT-PCR of CDK2 using mRNA obtained from the same samples. Data were normalized to the mean of controls values set to 1. *p ≤0.05, **p ≤0.01. f Schematic representation of METTL3/ADAR1 modulation in glioblastoma. METTL3/METTL14 methylates ADAR1 mRNA allowing the reader YTHDF1 to boost ADAR1 translation. The high level of ADAR1 protein (with unaltered ADAR1 mRNA) correlates with GBM patient OS and promotes cell proliferation by stabilizing CDK2. Moreover, the ablation of ADAR1 in an METTL3 unaltered background is sufficient to inhibit glioblastoma in vivo

Article Snippet: The antibodies used were as follows: ADAR1 (Santa Cruz Biotechnology), ADAR1 (Bethyl), CDK2 (Santa Cruz Biotechnology), YTHDF1 (Abcam), METTL3 (Abcam), METTL14 (Bethyl), cyclinE (Santa Cruz Biotechnology), p57 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), CDC14B (LifeSpan), ADAR2 (Santa Cruz Biotechnology), Ubiquitin (Thermo Fisher), β-actin (Santa Cruz Biotechnology), GAPDH (Cell Signaling), and the anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Injection, Generated, Control, Quantitative Proteomics, Staining, Quantitative RT-PCR, In Vivo

Journal: BMC Cancer

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

doi: 10.1186/s12885-022-10010-x

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

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

Techniques: Expressing, Staining

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: Specific ablation of Ythdf1 from dorsal commissural neurons results in decrease of Robo3.1 protein level. ( A ) Schematic drawings are shown for the genetic deletion strategy for Ythdf1 . Exon 4 which contains YTH domain-coding sequence is deleted after Cre-mediated recombination. ( B ) Depletion of YTHDF1 protein in the dorsal spinal cord of Wnt1-Cre +/- ;Ythdf1 fl/fl cKO mouse embryos. Anti YTHDF1 immunostaining of E11.5 spinal cord sections confirmed cKO of YTHDF1 protein from dorsal spinal cord and dorsal root ganglia (DRG), illustrated by asterisks. ( C ) Specific ablation of YTHDF1 protein from Atoh1-Cre + commissural neurons. Anti YTHDF1 immunostaining of E11.5 spinal cord sections confirmed cKO of YTHDF1 protein in YFP + commissural neurons in Atoh1-Cre +/ - ;Rosa26-YFP +/ - ;Ythdf1 fl/fl cKO mouse embryos, while YTHDF1 expression was intact in Atoh1-Cre +/ - ;Rosa26-YFP +/ - control embryos. ( D ) Ythdf1 cKO with Atoh1-Cre led to dramatic reduction of Robo3.1 protein from dorsal commissural axons. E10.5 pre-crossing DSC explants was dissected and cultured in vitro . Anti-Robo3.1 IF showed significant decline of Robo3.1 protein level in TAG1 (TSA)-positive commissural axons. Representative images are shown from eight Ythdf1 fl/fl and nine Atoh1-Cre +/ - ;Ythdf1 fl/fl embryos, respectively. ( E ) Quantification of Robo3.1 IF in commissural axons of cultured DSC explants from Ythdf1 cKO mouse embryos and their littermate controls. All data are mean ± S.E.M. and represented as box and whisker plots: Ythdf1 fl/fl ( n = 30 confocal fields) versus Atoh1-Cre +/ - ;Ythdf1 fl/fl ( n = 47 confocal fields), ** P = 0.0014; by unpaired Student's t test. Scale bars, 100 μm (B and D) and 10 μm (C).

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Sequencing, Immunostaining, Expressing, Control, Cell Culture, In Vitro, Whisker Assay

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: Robo3.1 mRNA is modified by m 6 A and bound by the m 6 A reader YTHDF1. ( A ) Predicted m 6 A sites in Robo3.1 mRNA by SRAMP program. ( B ) Anti-m 6 A IP pulled down Robo3.1 mRNA from RNA of mouse embryonic spinal cord using two different m 6 A antibodies (one mAb and another pAb) with corresponding IgG as controls. RT-PCR was performed to detect Robo3 .1 mRNA in elutes. ( C ) Verification of m 6 A sites in Robo3.1 mRNA. Anti-m 6 A IP failed to pull down Robo3.1 mRNA from COS-7 cells expressing Robo3.1 with m 6 A sites mutated ( Robo3.1-MT m6A ) compared with Robo3.1-WT . ( D ) Knockdown of METTL3 in commissural neurons. Dissociated commissural neurons from E10.5 mouse dorsal spinal cord was infected with lenti virus expressing shMettl3 , marked by eGFP labeling. Knockdown by shMettl3 for 48 h resulted in dramatic decrease of METTL3 protein levels in neurons, compared with shCtrl . ( E ) Knockdown of METTL3 led to significant decreases of Robo3.1 protein levels in commissural axons compared with shCtrl . Scale bar, 10 μm. ( F ) Quantification of relative Robo3.1 IF to eGFP in (E). All data are mean ± S.E.M. and are represented as box and whisker plots: **** P = 1.18E–5 ( n = 15 axons for shCtrl ; n = 19 axons for shMettl3 ), by unpaired Student's t test. ( G ) RNA IP (RIP) pulled down Robo3.1 mRNA from mouse embryonic spinal cord lysate with YTHDF1 antibody, but not with control IgG. ( H ) Binding of YTHDF1 with Robo3.1 mRNA is m 6 A-dependent. RIP using YTHDF1 antibody failed to pull down Robo3.1 mRNA from COS-7 cells co-expressing YTHDF1 and Robo3.1 with m 6 A sites mutated ( MT m6A ) compared with WT Robo3.1 .

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Modification, Reverse Transcription Polymerase Chain Reaction, Expressing, Knockdown, Infection, Virus, Labeling, Whisker Assay, Control, Binding Assay

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: The m 6 A reader YTHDF1 controls translation of Robo3.1 mRNA in an m 6 A-dependent manner. ( A ) YTHDF1 could enhance translation of Robo3.1 . Co-expression of WT Robo3.1 with YTHDF1 in COS-7 cells resulted in a dramatic increase of Robo3.1 protein level by IF, compared with an empty vector expressing eGFP only. However, YTHDF1 failed to increase translation of Robo3.1 with m 6 A sites mutated ( Robo3.1-MT m6A ). Scale bar, 25 μm. ( B ) Quantification of relative Robo3.1 IF to eGFP in (A). ( C ) Western blotting analysis showing regulation of Robo3.1 protein levels by YTHDF1 is through translational control. Protein synthesis inhibitor CHX blocked translation of Robo3.1 in HEK293T cells expressing HA-Robo3.1. Similar effects were found with cells co-expressing HA-Robo3.1 and YTHDF1. ( D ) Quantification of relative HA-Robo3.1 levels to β-actin in (C). All data are mean ± S.E.M. Data of IF quantification (B) are represented as box and whisker plots: *** P = 5.11E–04, ‘ Robo3.1-WT + IRES-eGFP ’ ( n = 16 cells) versus ‘ Robo3.1-WT + Ythdf1-IRES-eGFP ’ ( n = 17 cells); ns, not significant ( P = 0.41), ‘ Robo3.1-WT + IRES-eGFP ’ ( n = 15 cells) vs ‘ Robo3.1-MT m6A + Ythdf1-IRES-eGFP ’ ( n = 18 cells); by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. WB quantification data (D, n = 3 replicates) are represented as dot plots: ** P = 0.002 (‘HA-Robo3.1’ versus ‘HA-Robo3.1 + CHX’); ** P = 0.002 (‘HA-Robo3.1’ versus ‘HA-Robo3.1 + YTHDF1’); *** P = 1.41E-04 (‘HA-Robo3.1 + YTHDF1’ versus ‘HA-Robo3.1 + YTHDF1 + CHX’); by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Whisker Assay, Comparison

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: YTHDF1 regulates translation of endogenous Robo3.1 in commissural neurons and is controlled by floor plate. ( A ) Knockdown of YTHDF1 in commissural neurons. Dissociated commissural neurons from E10.5 mouse spinal cord was infected with lenti virus expressing shYthdf1-2 and shYthdf1-3 , respectively, and marked by eGFP labeling. Knockdown by shYthdf1 for 48 h resulted in dramatic decrease of YTHDF1 protein levels in commissural axons, compared with shCtrl . ( B ) Knockdown of YTHDF1 led to significant decreases of Robo3.1 protein levels in commissural axons compared with shCtrl . ( C ) Quantification of relative Robo3.1 IF to eGFP in (B). n = 16 axons for shCtrl ; n = 17 axons for shYthdf1-2 ; n = 16 axons for shYthdf1-3 . ( D ) Overexpression of YTHDF1 in commissural neurons. Dissociated commissural neurons from E10.5 mouse spinal cord was infected with lenti virus expressing YTHDF1, marked by eGFP labeling. Overexpression of YTHDF1 resulted in dramatic increase of YTHDF1 protein levels in commissural axons, compared with control. ( E ) Overexpression of YTHDF1 led to significant increases of Robo3.1 protein levels in commissural axons compared with eGFP control. ( F ) Quantification of relative Robo3.1 IF to eGFP in (E). n = 14 axons for IRES-eGFP ; n = 16 axons for Ythdf1-IRES-eGFP . ( G ) Regulation of YTHDF1 expression by floor plate. DSC explants from E10.5 mouse embryonic spinal cords were cultured with FP-CM or Ctrl-CM. WB analysis was carried out to measure YTHDF1 protein levels. ( H ) Quantification of WB signals in (G). All data are mean ± S.E.M. Data of IF quantification (C, F) are represented as box and whisker plots: For C, **** P = 8.05E-7 ( shYthdf1-2 versus shCtrl ), **** P = 3.83E-7 ( shYthdf1-3 versus shCtrl ), ns, not significant ( P = 0.76, shYthdf1-2 versus shYthdf1-3 ); for F, **** P = 3.35E–6; by unpaired Student's t test. WB quantification data (H, n = 5 replicates) are represented as dot plots: **** P = 1.68E–8; by paired Student's t test. Scale bars, 10 μm (B and E).

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Knockdown, Infection, Virus, Expressing, Labeling, Over Expression, Control, Cell Culture, Whisker Assay

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: Ythdf1 cKO embryos exhibit defects in pre-crossing commissural axon guidance. ( A ) Misprojection of pre-crossing commissural axons into motor columns in Ythdf1 cKO embryos. TAG1 marks commissural axons in E11.5 embryonic sections and there are significantly more misprojecting axons into motor columns (arrowheads) in Ythdf1 cKO embryos compared with their littermate controls. Representative images are shown from three Ythdf1 fl/fl and three Atoh1-Cre +/- ; Ythdf1 fl/fl embryos, respectively. ( B ) Quantification of commissural axons intruding into motor columns. All data are mean ± S.E.M. and represented as box and whisker plots: Ythdf1 fl/fl ( n = 15 sections) versus Atoh1-Cre +/- ; Ythdf1 fl/fl ( n = 12 sections), ** P = 0.0096; by unpaired Student's t test. ( C and D ) DiI tracing of commissural axons with open-book preparations. Pre-crossing axon guidance defects including stalling (arrowheads) and pre-crossing turning (arrows) were observed in Ythdf1 cKO embryos. Representative images of E10.5–11 (C) and E11.5 (D) were shown. ( E ) Quantification of phenotypes in (C and D). Total 31 DiI injections with four Ythdf1 fl/fl embryos and 40 DiI injections with three Atoh1-Cre +/- ; Ythdf1 fl/fl embryos were analyzed. Percentage of observations with phenotypes was calculated. Note that the summed percentage for Atoh1-Cre +/- ;Ythdf1 fl/fl is >100 because some of DiI injections were found with both stalling and pre-crossing turning phenotypes. Scale bars, 50 μm (A, C and D).

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Whisker Assay