Journal: Frontiers in immunology

Article Title: YTHDF1 Negatively Regulates Treponema pallidum -Induced Inflammation in THP-1 Macrophages by Promoting SOCS3 Translation in an m6A-Dependent Manner.

doi: 10.3389/fimmu.2022.857727

Figure Lengend Snippet: FIGURE 1 | m6A methylation: the m6A writer METTL3 and the reader YTHDF1 are upregulated in TP-infected macrophages. (A) Colorimetric quantification of m6A methylation in RNA from THP-1 cells with or without TP infection. (B, C) Western blot analysis of METTL3, METTL14, and YTHDF1 in THP-1 cells with or without TP infection. GAPDH was used as a control. *P < 0.05, **P < 0.01. (D, E) Immunofluorescence analysis of YTHDF1 or METTL3 in CD68+ macrophages in secondary syphilitic lesions and paired non-lesional skin tissue. Scale bar: 20 mm or 5 mm.

Article Snippet: For immunofluorescence analysis of the tissues, the sections were permeabilized with 0.25% Triton X-100 for 0.5 h and blocked with 5% goat serum for 1 h. Tissue sections were then incubated with primary antibodies against CD68 (Cat. No. 66231-2-Ig, Proteintech, Wuhan, Hubei, China) and YTHDF1 (Cat. No. 17479-1-AP, Proteintech) at 4°C overnight, followed by April 2022 | Volume 13 | Article 857727 incubation with the secondary antibody, Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) (A21206, Life Technologies) or Alexa Fluor® 594 donkey anti-mouse IgG (H+L) (A21203, Life Technologies).

Techniques: Methylation, Infection, Western Blot, Control

Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: The primers of qRT-PCR used in this study.

Article Snippet: Antibodies were used, including rabbit anti-YTHDF1 monoclonal antibody (1:1000，Cell signaling technology), mouse anti-GAPDH monoclonal antibody (1:1000,Abcam).

Techniques:

Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: Dexmedetomidine up-regulated the m6A level of HOTAIR in LPS-treated RAW264.7 cells. (A) The expression of HOTAIR m6A level was detect by MeRIP-qPCR. (B) HOTAIR was significantly enriched by YTHDF1 compared with IgG. (C) The expression of YTHDF1 was detect by qRT-PCR and WB in LPS-treated RAW264.7 cells. RAW264.7 cells were transfected with siYTHDF1 (300 nM) or pYTHDF1 (10 μg) in a 100-mm dish, and the cell lysate was divided into three parts: one for WB to determine the expression of YTHDF1 (D), and the other two for qRT-PCR to assess HOTAIR expression (E) and MeRIP-qPCR to determine the m6A level on HOTAIR (F).

Article Snippet: Antibodies were used, including rabbit anti-YTHDF1 monoclonal antibody (1:1000，Cell signaling technology), mouse anti-GAPDH monoclonal antibody (1:1000,Abcam).

Techniques: Expressing, Quantitative RT-PCR, Transfection

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

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

Figure Lengend Snippet: Figure 1. 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 (log2(fold change) < 0.5; log10(P value < 1). Middle, top enriched genes in shYTHDF1 cells vs. shControl (log2(fold change) > 0.5; log10(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 ¼ 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 ¼ 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 χ2 test (G), or log rank test (H and I).

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

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

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

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

Figure Lengend Snippet: Figure 2. 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 ¼ 10; A), colony formation (N ¼ 3, 7–14 days; B), apoptosis (N ¼ 3; C), and G1-S cell cycle transition (N ¼ 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 ¼ 5; HCT116, N ¼ 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: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

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 m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

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

Figure Lengend Snippet: Figure 3. YTHDF1 directly targets m6A-modified RUVBL1/2 mRNA methylation and promotes their protein expression in vitro and in vivo. A, UCSC snapshots of m6A-seq reads of RUVBL1/2 mRNA in DLD1 cells. The normalized read densities are shown for m6A (orange) and input (blue). B, Methylated RIP-qPCR analysis of m6A-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 (Ythdf1lslCdx2-CreERT2) as compared with wildtype mice. Student t test (B–D) and one-way ANOVA (E). ****, P< 0.0001.

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

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 m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

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

Figure Lengend Snippet: Figure 4. 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 30UTR sequences, followed by luciferase assays. E, pmirGLO-RUVBL1/2-mutant reporters with mutated m6A sites (RRACH to TTTCT) in the 30UTR 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 immu- nofluorescence 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: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

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

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

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

Figure Lengend Snippet: Figure 5. 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: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

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

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

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

Figure Lengend Snippet: Figure 6. 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-IC50 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: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: Over Expression, Positive Control, Expressing, Mutagenesis

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

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

Figure Lengend Snippet: Figure 7. 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: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

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

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: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: KD of YTHDF1 or YTHDF2 significantly promoted GC axon growth in vitro. A) Representative confocal images showing YTHDF1 and YTHDF2 are expressed in the growth cones and axons of cultured P6–P8 GCs. B) Western blotting (WB) validating the KD efficiency of shYthdf1 in cultured GCs. Data of quantification are mean ± SEM and represented as dot plots ( n = 3): shYthdf1#2 versus shCtrl , **** p = 3.91E‐08; shYthdf1#3 versus shCtrl , **** p = 8.62E‐09; by one‐way ANOVA followed by Tukey's multiple comparison test. C) WB validating the KD efficiency of shYthdf2 in cultured GCs. Data of quantification are mean ± SEM and represented as dot plots ( n = 3): shYthdf2#1 versus shCtrl , **** p = 9.15E‐06; shYthdf2#3 versus shCtrl , **** p = 2.53E‐06; by one‐way ANOVA followed by Tukey's multiple comparison test. D) Representative images showing that axon growth rates of GCs are significantly increased after KD of YTHDF1. Black and blue arrowheads indicate the terminals of the same axons imaged at 0 and 15 h, respectively. E) Quantification of axon growth rates in (D). Data are represented as box and whisker plots: shYthdf1#2 versus shCtrl , ** p = 0.0030; shYthdf1#3 versus shCtrl , *** p = 1.39E‐04; n = 24 axons for each group; by one‐way ANOVA followed by Tukey's multiple comparison test. F) Representative images showing that axon growth rates of GCs are significantly increased after KD of YTHDF2. Black and blue arrowheads indicate the terminals of the same axons imaged at 0 and 15 h, respectively. G) Quantification of axon growth rate in (F). Data are represented as box and whisker plots: shYthdf2#1 versus shCtrl , **** p = 1.40E‐07; shYthdf2#3 versus shCtrl , **** p = 6.38E‐08; n = 24 axons for each group; by one‐way ANOVA followed by Tukey's multiple comparison test. A,D,F) Scale bars represent 10 µm.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques: In Vitro, Cell Culture, Western Blot, Comparison, Whisker Assay

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: The putative mRNA targets were identified by anti‐YTHDF1 and anti‐YTHDF2 RIP‐seq. A) Venn diagram showing numbers of mRNA targets identified by anti‐YTHDF1 and anti‐YTHDF2 RIP‐seq. B,C) GO analysis of target mRNAs identified by B) anti‐YTHDF1 and C) anti‐YTHDF2 RIP‐seq. The GO terms in Biological Process are shown. The most relevant terms are highlighted in red texts. D,E) KEGG analysis of target mRNAs identified by D) anti‐YTHDF1 and E) anti‐YTHDF2 RIP‐seq. Axon guidance, mTOR signaling pathway, and Wnt signaling pathway are highlighted in red texts.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques:

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: The differentially expressed genes were identified by proteome and transcriptome analysis after KD of YTHDF1 and YTHDF2, respectively. A) GO analysis of downregulated proteins revealed by quantitative proteomic analysis after YTHDF1 KD in GCs. B) KEGG analysis of downregulated proteins revealed by quantitative proteomic analysis. The Wnt signaling pathway is highlighted in red texts. C) Heatmap showing the differential expression profiling of genes by RNA‐seq after YTHDF2 KD in GCs. D) GO analysis of differentially expressed genes revealed by RNA‐seq after YTHDF2 KD in GCs. E) Axon‐related GO terms of differentially expressed genes revealed by RNA‐seq after YTHDF2 KD in GCs. The GO terms in Biological Process are shown. BP, biological process; MF, molecular function; CC, cellular component.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques: Quantitative Proteomics, RNA Sequencing

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: YTHDF1 and YTHDF2 regulate local translation of Dvl1 and Wnt5a , respectively, to control the GC axon growth. A) Relative Dvl1 protein level detected by TMT‐labeled proteomic analysis after YTHDF1 KD. Data are mean ± SEM: * p = 0.047; n = 3 replicates; by unpaired Student's t test. B) RT‐qPCR confirming the Dvl1 mRNA level was unchanged after KD of YTHDF1 in GCs. Data are mean ± SEM: p = 0.78; n = 3; ns, not significant; by unpaired Student's t test. C) Relative Wnt5a mRNA level measured by RNA‐seq after YTHDF2 KD. Data are mean ± SEM: **** p = 4.92E‐05; n = 3 replicates; by unpaired Student's t test. D) Axon growth rate significantly increased after KD of Dvl1. Quantification of axon growth rates after KD of Dvl1 using siRNAs. Data are represented as box and whisker plots: n = 21 axons for each group; siDvl1#4 versus siCtrl , **** p = 2.09E‐05; siDvl1#5 versus siCtrl , **** p = 3.15E‐10. All by one‐way ANOVA followed by Tukey's multiple comparison test. E) Axon growth rate significantly decreased after KD of Wnt5a. Quantification of axon growth rates after KD of Wnt5a using siRNAs. Data are represented as box and whisker plots: n = 20 axons for each group; siWnt5a#1 versus siCtrl , **** p = 4.79E‐05; siWnt5a#3 versus siCtrl , **** p = 4.96E‐06. All by one‐way ANOVA followed by Tukey's multiple comparison test. F) Dvl1 and Wnt5a mRNAs were detected in axons by RT‐PCR using total RNA from pure axons or soma, respectively. Similar to β‐actin mRNA which is a positive control for axonal mRNAs, Dvl1 and Wnt5a mRNAs were detected in both axons and soma. The absence of H1f0 mRNA from axons indicated that the axonal material was pure with no soma incorporation. G,H) Detection of Dvl1 and Wnt5a mRNA localization in growth cones of GC neurons by FISH. Dissociated GCs were cultured for 2 DIV and then FISH was performed using RNAscope riboprobes. Dvl1 and Wnt5a mRNAs were detected in growth cones of GC neurons as red punctate patterns. β‐actin and Dapb serve as positive and negative controls, respectively. Tuj1 immunostaining was used to visualize axons. Quantification of puncta density was shown in (H). I,J) Compartmentalized KD of Dvl1 in GC axons. GCs were cultured in microfluidic chambers and siDvl1 was specifically transfected to axons only. Compared with siCtrl, siDvl1#4 and siDvl1#5 led to significant decrease of Dvl1 IF signals. Quantification data are represented as box and whisker plots (J). siDvl1#4 ( n = 15 axons) versus siCtrl ( n = 18 axons), **** p = 1.11E‐11; siDvl1#5 ( n = 17 axons) versus siCtrl , **** p = 3.56E‐12; by one‐way ANOVA followed by Tukey's multiple comparison test. K) Axon growth rates significantly increased after axon‐specific KD of Dvl1. Data are represented as box and whisker plots. siDvl1#4 ( n = 18 axons) versus siCtrl ( n = 21 axons), ** p = 0.0024; siDvl1#5 ( n = 17 axons) versus siCtrl , *** p = 0.00049; by one‐way ANOVA followed by Tukey's multiple comparison test. L,M) Compartmentalized KD of Wnt5a in axons. Compared with siCtrl , siWnt5a#1 and siWnt5a#3 led to significant decrease of Wnt5a IF signals. Quantification data are represented as box and whisker plots (M). siWnt5a#1 ( n = 38 axons) versus siCtrl ( n = 32 axons), **** p = 6.80E‐14; siWnt5a#3 ( n = 39 axons) versus siCtrl , **** p = 5.20E‐14; by one‐way ANOVA followed by Tukey's multiple comparison test. N) Axon growth rates significantly decreased after axon‐specific KD of Wnt5a which can be rescued by application of recombinant Wnt5a protein into axonal compartments. Data are represented as box and whisker plots. siWnt5a#1 ( n = 15 axons) versus siCtrl ( n = 16 axons), **** p = 3.86E‐06; siWnt5a#3 ( n = 19 axons) versus siCtrl , **** p = 1.59E‐07; siWnt5a#1 +rWnt5a ( n = 18 axons) versus siCtrl , p = 0.34; siWnt5a#3 +rWnt5a ( n = 16 axons) versus siCtrl , p = 0.85; siWnt5a#1 +rWnt5a versus siWnt5a#1 , * p = 0.036; siWnt5a#3 +rWnt5a versus siWnt5a#3 , **** p = 3.40E‐05; ns, not significant; by one‐way ANOVA followed by Tukey's multiple comparison test. O) Overexpression of YTHDF1 increased axonal Dvl1 protein level in cultured GCs and axon‐specific siDvl1 KD eliminated this increase. Data are represented as box and whisker plots. Ythdf1‐IRES‐GFP + siCtrl versus IRES‐GFP + siCtrl , **** p = 1.34E‐05; Ythdf1‐IRES‐GFP + siDvl1#4 versus IRES‐GFP + siDvl1#4 , p = 0.99; Ythdf1‐IRES‐GFP + siDvl1#5 versus IRES‐GFP + siDvl1#5 , p = 0.84; IRES‐GFP + siDvl1#4 versus IRES‐GFP + siCtrl , p = 5.60E‐14; IRES‐GFP + siDvl1#5 versus IRES‐GFP + siCtrl , p = 5.80E‐14; Ythdf1‐IRES‐GFP + siDvl1#4 versus Ythdf1‐IRES‐GFP + siCtrl , **** p = 1.01E‐15; Ythdf1‐IRES‐GFP + siDvl1#5 versus Ythdf1‐IRES‐GFP + siCtrl , **** p = 1.02E‐15; ns, not significant; n = 27 axons for each group; by one‐way ANOVA followed by Tukey's multiple comparison test. P) KD of YTHDF2 increased axonal Wnt5a protein level in GCs and axon‐specific siWnt5a KD eliminated this increase. Data are represented as box and whisker plots. shYthdf2#3 + siCtrl versus shCtrl + siCtrl , **** p = 4.30E‐15; shYthdf2#3 + siWnt5a#1 versus shCtrl + siWnt5a#1 , p = 0.99; shYthdf2#3 + siWnt5a#3 versus shCtrl + siWnt5a#3 , p = 0.89; shCtrl + siWnt5a#1 versus shCtrl + siCtrl , **** p = 1.34E‐11; shCtrl + siWnt5a#3 versus shCtrl + siCtrl , p = 1.01E‐13; shYthdf2#3 + siWnt5a#1 versus shYthdf2#3 + siCtrl , **** p = 1.01E‐15; shYthdf2#3 + siWnt5a#3 versus shYthdf2#3 + siCtrl , **** p = 1.02E‐15; ns, not significant; n = 27 axons for each group; by one‐way ANOVA followed by Tukey's multiple comparison test. Scale bars represent G) 10 µm and I,L) 5 µm.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques: Control, Labeling, Quantitative RT-PCR, RNA Sequencing, Whisker Assay, Comparison, Reverse Transcription Polymerase Chain Reaction, Positive Control, Cell Culture, RNAscope, Immunostaining, Transfection, Recombinant, Over Expression

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: Parallel fiber growth was enhanced in both Ythdf1 and Ythdf2 cKO mice. A,B) Representative images of A) YTHDF1 and B) YTHDF2 immunostaining in P15 cerebellum of A) Ythdf1 and B) Ythdf2 cKO, respectively. YTHDF1 or YTHDF2 was successfully eliminated in GCs while their expression in PCs was not affected. Scale bars represent 500 µm. C,D) Lengths of parallel fibers labeled by DiI were significantly increased in Ythdf1 and Ythdf2 cKO mice. The white arrowheads indicate the terminals of DiI‐labeled PFs. Scale bars represent 100 µm. E,F) Quantification of parallel fiber (PF) lengths in (C) and (D). Data are expressed as box and whisker plots. In (E), **** p = 1.38E‐05; for Ythdf1 fl/fl mice, n = 36 confocal fields from 11 pups, for Ythdf1 cKO mice, n = 42 confocal fields from 11 pups. In (F), **** p = 2.29E‐05; for Ythdf2 fl/fl mice, n = 46 confocal fields from 13 pups, for Ythdf2 cKO mice, n = 43 confocal fields from 12 pups. All by unpaired Student's t test. G,H) Significantly higher Tag1 IF in the deep layer of cerebellar EGL of Ythdf1 and Ythdf2 cKO mice was detected. Scale bars represent 40 µm. I,J) Quantification of Tag1 IF intensity signals in (G) and (H). Data are expressed as box and whisker plots. In (I), **** p = 2.80E‐06; n = 18 confocal fields for Ythdf1 fl/fl mice, n = 15 confocal fields for Ythdf1 cKO mice. In (J), **** p = 2.02E‐12; n = 26 confocal fields for Ythdf2 fl/fl mice, n = 28 confocal fields for Ythdf2 cKO mice. All by unpaired Student's t test.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques: Immunostaining, Expressing, Labeling, Whisker Assay

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: Synapse formation was promoted in Ythdf1 and Ythdf2 cKO cerebella. A–E) Representative immunoblots showing that the protein levels of synaptic markers GluR δ 2, Nrxn1, and PSD95 were increased in A) Ythdf1 cKO cerebellum at P30. Quantification of B) YTHDF1, C) GluR δ 2, D) Nrxn1, and E) PSD95. For (B), *** p = 0.00064; for (C), * p = 0.016; for (D), * p = 0.028; for (E), * p = 0.013; n = 3 replicates; by unpaired Student's t test. F–J) Representative immunoblots showing that GluR δ 2, Nrxn1, and PSD95 protein levels were increased in F) Ythdf2 cKO cerebellum at P30. Quantification of G) YTHDF2, H) GluR δ 2, I) Nrxn1, and J) PSD95. For (G), ** p = 0.0037; for (H), **** p = 2.65E‐05; for (I), ** p = 0.0062; for (J), * p = 0.028; n = 3 replicates; by unpaired Student's t test. K–N) Representative images of VGLUT1 and PSD95 co‐immunostaining in the ML of P30 cerebellum of K) Ythdf1 and M) Ythdf2 cKO. VGLUT1 + /PSD95 + puncta were counted to measure the number of synapses and quantifications were shown in (L) and (N). Data are expressed as box and whisker plots. In (L), *** p = 1.10E‐04; in (N), **** p = 2.16E‐07; n = 20 confocal fields for each group; by unpaired Student's t test. Scale bars represent K,M) 5 µm.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques: Western Blot, Immunostaining, Whisker Assay

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: The motor coordination ability is enhanced in Ythdf1 and Ythdf2 cKO mice. A) Ythdf1 and B) Ythdf2 cKO showed normal animal size and cerebellar development at P40. Scale bars in the upper panels represent 1 cm and scale bars in the lower panels represent 0.25 cm. C,D) Normal body weight in Ythdf1 and Ythdf2 cKO mice. In (C), p = 0.99; n = 15 for Ythdf1 fl/fl mice; n = 14 for Ythdf1 cKO mice. In (D), p = 0.37; n = 9 for Ythdf2 fl/fl mice; n = 10 for Ythdf2 cKO mice; ns, not significant. All by unpaired Student's t test. E,F) The latency to fall measurements for E) each and F) total trial in rotarod test of Ythdf1 cKO mice. In (E), for Day1‐Run #3, * p = 0.037; for Day2‐Run #1, ** p = 0.0049; for Day3‐Run #1, * p = 0.039; for Day3‐Run #2, * p = 0.034; for Day3‐Run #3, * p = 0.037. In (F), * p = 0.027; n = 15 for Ythdf1 fl/fl mice; n = 14 for Ythdf1 cKO mice. All by unpaired Student's t test. G,H) The latency to fall measurements for G) each and H) total trial in rotarod test of Ythdf2 cKO mice. In (G), for Day1‐Run #3, ** p = 0.0020; for Day2‐Run #3, ** p = 0.0089; for Day3‐Run #1, * p = 0.038; for Day3‐Run #2, * p = 0.013. In (H), * p = 0.015; n = 15 for Ythdf2 fl/fl mice; n = 14 for Ythdf2 cKO mice. All by unpaired Student's t test.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques:

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: A working model shows that YTHDF1 and YTHDF2 work synergistically to regulate Wnt5a‐PCP signaling pathway and cerebellar granule cell axon growth. A) Under normal conditions, YTHDF1 promotes the translation of m 6 A‐modified Dvl1 mRNA in GC axons. Dvl1 can block Wnt5a‐Fzd3‐activated PCP signaling. Meanwhile, YTHDF2 facilitates Wnt5a mRNA degradation to downregulate Wnt5a protein level in GC axons. So both YTHDF1 and YTHDF2 negatively regulate Wnt5a‐PCP signaling pathway and GC axon growth. B) In Ythdf1 and Ythdf2 cKO mice, local translation of Dvl1 mRNA and decay of Wnt5a mRNA in GC axons are inhibited, respectively. The resulting downregulation of Dvl1 and upregulation of Wnt5a protein levels in axons potentiate Wnt5a‐PCP signaling and promote GC axon growth.

Article Snippet: Then sections were incubated with primary antibodies diluted in blocking solution overnight at 4 °C: YTHDF1 (1:500), YTHDF2 (1:500), NeuN (1:500, 24307, Cell Signaling Technology), VGLUT1 (1:10 000, 135303, Synaptic Systems), PSD95 (1:100, ab2723, Abcam).

Techniques: Modification, Blocking Assay

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