Journal: Biochemical pharmacology

Article Title: m 6 A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2.

doi: 10.1016/j.bcp.2021.114766

Figure Lengend Snippet: Fig. 4. Posttranscriptional regulation of CES2 by RNA methylation via m6A reader protein YTHDC2. The stability of CES2 mRNA in siRNA-transfected HepG2 cells was examined (A-C). HepG2 cells were treated with 10 ng/µL α-amanitin 48 h after transfection with siMETTL3 and siMETTL14 (A), siFTO (B), siALKBH5 (C), or siControl (A-C). Total RNA was prepared after 0, 12, 24, and 36 h. The CES2 mRNA level was determined by using real-time RT-PCR. The CES2 mRNA levels at time 0 (the time of addition of α-amanitin) in each treatment were assigned values of 100%. YTHDC2 mRNA, YTHDF2 mRNA (D), CES2 mRNA (E), and CES2 protein (F) levels in siYTHDC2- or siYTHDF2-transfected HepG2 cells were determined by real-time RT-PCR and Western blotting. The mRNA and protein levels were normalized to β-actin levels. The values represent the levels relative to siControl. (G) Cell lysates from HepG2 cells were immunoprecipitated with an anti-human YTHDC2 antibody or normal rabbit IgG. An electropherogram of the PCR amplicon using primers for CES2 mRNA is shown. The length of the PCR product was 316 bp. Each point and column represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.01 compared with siControl.

Article Snippet: The rabbit anti-human YTHDC2 polyclonal antibody was from Bethyl Laboratories (Montgomery, TX).

Techniques: Methylation, Transfection, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Amplification

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet:

Article Snippet: Rabbit anti-YTHDC2 , Novus Biologicals , Cat# NBP1–85089; RRID: AB_11034900.

Techniques: Recombinant, Software

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Expression profiles and characterization of circYthdc2. A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Expressing, Selection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Negative Control, Nucleic Acid Electrophoresis

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: CircYthdc2 encodes a 170 amino acid (aa) novel protein, Ythdc2-170aa. A Upper panel, the putative ORF in circYthdc2. Lower panel, the sequences of the putative ORF are shown. B The putative IRES activity in circYthdc2 was tested. C Left panel: Full-length or truncated circYthdc2 IRES sequences were cloned before GFP as indicated to construct reporter plasmids. Right panel: The empty vector, and full-length or truncated IRES vector were cotransfected with si-eif4E into HEK293 cells, and GFP signals were detected. D Schematic diagram of FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmid construction. E Upper panel: The putative Ythdc2-170aa amino acid sequences and antibody generation region were shown as indicated to produce the Ythdc2-170aa antibody. The red amino acids were distinctly formed by the circYthdc2 junction. Lower: FLAG tag antibody was used to detect Ythdc2-170aa expression in MKC cells transfected with the vectors mentioned in Fig. 2D. In addition, Ythdc2-170aa antibody was used to detect Ythdc2-170aa expression in MKC cells after SCRV infection F FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmids were transfected into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2-170aa cellular localization. Original magnification is 630; all data represent the means ± SE from three independent triplicate experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Activity Assay, Clone Assay, Construct, Plasmid Preparation, FLAG-tag, Expressing, Transfection, Infection, Immunofluorescence, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: CircYthdc2 and Ythdc2-170aa inhibit host antiviral innate immunity. A and B The schematic diagram of siRNAs ( A ) and circYthdc2 overexpression plasmid structure ( B ). C qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MIC cells treated with siRNAs. qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MKC cells overexpressing circYthdc2. D and E qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-circYthdc2 ( D ) and transfected in MKC cells with circYthdc2 or pLC5-circ and Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG after SCRV infection ( E ). F circYthdc2 and Ythdc2-170aa promote SCRV replication. MIC cells transfected with NC or si-circYthdc2 and MKC cells were transfected with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. G MKC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MKC cells were transfected with Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG. H Effect of circYthdc2 on cell viability after SCRV infection. MIC cells were transfected with NC or si-circYthdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay was measured. I Ythdc2-170aa counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING, MAVS, TRIF, and TBK1 expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa. J Relative protein levels of STING in MIC cells after transfected with NC or si-circYthdc2 and in MKC cells with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa or Linear-FL-Ythdc2-AG. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Over Expression, Plasmid Preparation, Expressing, Transfection, Infection, RNA Expression, Staining, Viability Assay, Luciferase, Cotransfection

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Ythdc2 inhibits host antiviral innate immunity. A Relative protein and mRNA levels of Ythdc2 in MIC cells after transfected with NC or si-circYthdc2. B Relative protein levels of STING in MIC cells after transfected with NC or si-Ythdc2 and in MKC cells with pcDNA3.1 or Ythdc2. C and D qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-Ythdc2 ( C ) and transfected in MKC cells with pcDNA3.1 and Ythdc2 after SCRV infection ( D ). E Ythdc2 counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1, Ythdc2. F Ythdc2 promotes SCRV replication. MIC cells transfected with NC or si-Ythdc2 and MKC cells were transfected with pcDNA3.1 or Ythdc2 for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. (G) MIC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MIC cells were transfected with NC or si-Ythdc2. H Effect of Ythdc2 on cell viability after SCRV infection. MIC cells was transfected with NC or si-Ythdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay were measured. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Transfection, Expressing, Infection, Luciferase, Cotransfection, Plasmid Preparation, RNA Expression, Staining, Viability Assay

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. A MKC cells were transfected with Flag-Ythdc2 and Flag-circYthdc2 plasmids, the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed; MIC cells were silence Ythdc2 or circYthdc2, and the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed B MKC cells were transfected with Linear-Flag-Ythdc2-170aa or Flag-circYthdc2 or Flag-Ythdc2 plasmids, after 42 h, the cells were treated with DMSO or 10 μM MG132 for 6 h before immunoblot analysis was performed. C Flag-Ythdc2 and Linear-Flag-Ythdc2-170aa were cotransfected with GFP-STING into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2 and STING or Ythdc2-170aa and STING cellular localization. Original magnification is 630. D Immunoprecipitation and immunoblot analysis of Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa with Myc-STING, in EPC cells. IP, immunoprecipitation. E Upper panel: Schematic diagram of Ythdc2-△HELICc plasmid construction. Lower panel: Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, Flag-Ythdc2-△HELICc or Linear-Flag-Ythdc2-170aa with Myc-STING, in MKC cells. IP, immunoprecipitation. F Schematic diagram of STING, STING-△TM, STING-△N, STING-△C plasmid construction. G Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. H Immunoprecipitation and immunoblot analysis of Linear-Flag-Ythdc2-170aa with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. I Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. J Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2, or Flag-Ythdc2-△HELICc, or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. K Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the three independent triplicated experiments

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Plasmid Preparation, Control, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: N6-methyladenosine modification mediates circYthdc2 translation polypeptides. A Upper panel: Schematic diagram of Flag-circYthdc2-m 6 A-mut plasmid construction. Lower panel: MKC cells were transfected with Flag-circYthdc2 or Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. B Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with m 6 A modification-related genes, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. C Left panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with METTL3 or METTL14, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Middle panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with FTO or YTHDF1, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Right panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with YTHDF3 or Ythdc2, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. D Relative RNA levels of circYthdc2 in MKC cells after transfected with pcDNA3.1, METTL3, METTL14, YTHDF1, YTHDF3, FTO, and Ythdc2, respectively. E The m 6 A level alteration of circYthdc2 upon METTL3 or FTO overexpression was examined by MeRIP-qPCR. MKC cells were transfected with vector or METTL3 or FTO plasmid for 48 h. F The level of circYthdc2 upon YTHDF1 or YTHDF3 or Ythdc2 overexpression were examined by RIP-qPCR. MKC cells were transfected with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. G The protein level of YTHDF1 or YTHDF3 or Ythdc2 was examined by RNA pulldown. MKC cells were transfected MS2-GFP, MS2-circYthdc2 or MS2-circYthdc2-m 6 A-mut with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. H Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, circYthdc2, METTL3, METTL14, FTO, YTHDF1, YTHDF3. I Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-circYthdc2 or m 6 A modification-related genes expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Modification, Plasmid Preparation, Transfection, Western Blot, Over Expression, Luciferase, Cotransfection, Expressing, Ubiquitin Proteomics

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Ythdc2-170aa is highly conserved in structure and function in vertebrates. A CircYthdc2 exists in Miichthys miiuy , Nibea albiflora , Sciaenops ocellatus , Larimichthys ocellatus , Xenopus tropicalis (GenBank accession no. XM_031893156.1), Bufo gargarizans (GenBank accession no. XM_044275926.1), Podarcis muralis (GenBank accession no. XM_028748342.1), Numida meleagris (GenBank accession no. XM_021380759.1), Mus musculus (GenBank accession no. NM_001163013.1), Homo sapiens (GenBank accession no. NM_022828.5), and is composed of exon 13 to exon 18, with a length of 634nt. We confirmed the head-to-tail splicing of hsa -circYthdc2 in the hsa -circYthdc2 RT-PCR product by Sanger sequencing. B Sequence alignment of circYthdc2 from teleost fish to mammals. C Amino acid sequence alignment of circYthdc2 translated polypeptides from teleost fish to mammals. D HEK293 cells were transfected with vector or hsa -Flag-circYthdc2, hsa -Flag-circYthdc2-ATG-mut, hsa -Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. E HEK293 cells were transfected STING and hsa -Flag-circYthdc2 and hsa -Flag-circYthdc2-m 6 A-mut with METTL3 or METTL14 or YTHDF1 or FTO plasmids, after 48 h, the immunoblot analysis was performed. F HEK293 cells were transfected STING and hsa -Flag-circYthdc2 with si- has -METTL3 or si- has -METTL14 or si- has -YTHDF1 or si- has -FTO, after 48 h, the immunoblot analysis was performed. All data represented the three independent triplicated experiments

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Plasmid Preparation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Schematic diagram of the mechanism underlying Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. Under normal circumstances, circYthdc2 does not translate to produce polypeptides. When SCRV virus infects the host, the pathway of circYthdc2 translating polypeptides is activated. There are two pathways for circYthdc2 to be translated into polypeptides, the one is IRES-mediated translation pathway and another m 6 A modification mediated translation pathway. In addition, Ythdc2 will preferentially promote the RNA degradation of circYthdc2 when circYthdc2 is produced in large quantities. Ythdc2-170aa and Ythdc2 both could promote the STING protein degradation and represses STING-mediated antiviral responses, thereby regulating viral replication. Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING, thereby inhibited the antiviral responses and help the virus escape

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Ubiquitin Proteomics, Virus, Modification, Produced

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Ythdc2 as a direct target of zinc finger protein 36 (Zfp36) is upregulated in cardiac hypertrophy. (A) Gene Ontology (GO) analysis used the potential targets genes of Zfp36 and also exhibited high expression levels in RNA sequencing (RNA‐seq) data. (B‒E) qRT‐PCR analysed the mRNA expression levels of the potential targets of Zfp36 which were enriched in m 6 A regulating enzymes pathway ( n = 3‒5). (F) Zfp36 protein structure diagram and the predicted binding region with Ythdc2 and docking results of Zfp36 protein with Ythdc2 3′ untranslated region (3′UTR) molecule, green chain (Ythdc2 3′UTR), the blue chain (Zfp36), the rod‐like structure represents the interacting amino acids and nucleic acids. (G) RNA‐pull down analysed the binding relation between Zfp36 and Ythdc2 mRNA. (H) RNA immunoprecipitation (RIP)‐PCR analysed the binding relation between Zfp36 and Ythdc2 mRNA ( n = 3). (I) Co‐localisation of Zfp36 and Ythdc2 mRNA in cardiomyocyte. Zfp36 identified with Zfp36 antibody (green), Ythdc2 mRNA identified by its probes (red) and nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) (blue). (J) Luciferase reporters of Ythdc2 3′UTR and ACTB 3′UTR in Hek293T cells transfected with increasing Zfp36 ( n = 3). (K) qRT‐PCR analysed the mRNA expression levels of Ythdc2 treated with or not Act D in over‐expression or knockdown Zfp36 ( n = 3‒6). (L and M) Over‐expression or knockdown Zfp36 CM was transfected with Ythdc2 3′UTR or ACTB 3′UTR luciferase plasmids, then subjected to analysis the expression level of luciferase mRNA at indicated time points treated with or not Act D ( n = 4). (N) RIP‐PCR analysed the binding relation between Zfp36 and Ythdc2 mRNA ( n = 3). (O and P) Real‐time PCR and western blotting analysed the expression levels of Ythdc2 ( n = 6). (Q) DCFH‐DA probe staining for the reactive oxygen species (ROS) levels of cardiomyocytes ( n = 6). (R) Detection of malondialdehyde (MDA) for lipid peroxidation level ( n = 5). (S) Western blot results shown the protein expression level Gpx4 ( n = 6). (T) The representative photographs of cardiomyocytes identified with α‐actinin and averaged data of cell area ( n = 11‒12). (U) The protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 6). Statistical analysis was performed with Student's t ‐test or one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Binding Assay, RNA Immunoprecipitation, Staining, Luciferase, Transfection, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Knockdown Ythdc2‐attenuated ferroptosis in hypertrophy induced by transverse aortic constriction (TAC). (A and K) Detection of malondialdehyde (MDA) for lipid peroxidation level ( n = 4‒5). (B and L) Western blot results show the protein expression level of Gpx4 ( n = 6). (C) Representative images of transmission electron microscope in mice. (D) Representative images of echocardiography recording, heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (E) Left ventricular posterior wall thickness dimensions during diastole in indicated mice ( n = 7‒9). (F and G) Heart weight‐to‐body weight ratio and heart weight‐to‐tibia length ratio ( n = 6‒8). (H) Quantification of myocyte area from WGA staining heart sections ( n = 5). (I and N) Western blot results shown the protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 6). (J) DCFH‐DA probe staining for the reactive oxygen species (ROS) levels of cardiomyocytes ( n = 6). (M) Representative photographs and averaged data of cell area stained by α‐actinin antibody the averaged data of cell area ( n = 10‒14). Statistical analysis was performed with one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Knockdown, Western Blot, Expressing, Transmission Assay, Microscopy, Staining, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Ythdc2 exacerbates ferroptosis and cardiac hypertrophy. (A and J) Detection of malondialdehyde (MDA) for lipid peroxidation level ( n = 5‒6). (B and K) Western blot results shown the protein expression level Gpx4 ( n = 5). (C) Representative images of echocardiography recording, heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (D) Left ventricular posterior wall thickness dimensions during diastole in indicated mice ( n = 6‒7). (E and F) Heart weight‐to‐body weight ratio and heart weight‐to‐tibia length ratio ( n = 5‒7). (G) Quantification of myocyte area from WGA staining heart sections ( n = 5). (H and M) Western blot results show the protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 5). (I) DCFH‐DA probe staining for the reactive oxygen species (ROS) levels of cardiomyocytes ( n = 6). (L) The representative photographs and averaged data of cell area stained by α‐actinin antibody (red), nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) (blue) ( n = 13‒16). Statistical analysis was performed with one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Western Blot, Expressing, Staining, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Regulated of Ythdc2 expression by zinc finger protein 36 (Zfp36) mediates the ferroptosis and cardiac hypertrophy phenotypes. (A and I) Detection of malondialdehyde (MDA) for lipid peroxidation level in mice hearts ( n = 5‒7). (B and J) Western blot results shown the protein expression level Gpx4 ( n = 5‒6). (C and K) Representative images of heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (D and L) Left ventricular (LV) posterior wall thickness dimensions in indicated mice ( n = 5‒7). (E and M) Heart weight‐to‐body weight ratio ( n = 6‒7). (F and N) Heart weight‐to‐tibia length ratio ( n = 5‒7). (G and O) Quantification of myocyte area from WGA stained ( n = 5). (H and P) Protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 5‒6). Statistical analysis was performed with one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Expressing, Western Blot, Staining, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Zinc finger protein 36 (Zfp36) through the Ythdc2/SLC7A11/glutathione (GSH)‐dependent ferroptosis pathway improve cardiac hypertrophy. (A‒H) The mRNA and protein expression levels of SLC7A11 ( n = 4‒5). (I) RNA immunoprecipitation (RIP)‐PCR analysed the m 6 A modification of SLC7A11 mRNA ( n = 3). (J) RNA‐pull down verified Ythdc2 bond to SLC7A11 mRNA ( n = 3). (K) RIP analysed the binding relation between Ythdc2 and SLC7A11 mRNA ( n = 3). (L) Co‐localisation of Ythdc2 and SLC7A11 mRNA in cardiomyocyte. (M) Real‐time PCR analysed the mRNA expression levels of SLC7A11 treated with or not Act D in over‐expression or knockdown Ythdc2 ( n = 3‒4). (N‒Q) Protein expression levels of SLC7A11 in mice hearts ( n = 4‒6). (R and S) GSH was negatively regulated by Ythdc2 in cardiac hypertrophy ( n = 3). (T‒U) Zfp36 regulates GSH level by Ythdc2 ( n = 3‒4). Statistical analysis was performed with Student's t ‐test or one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Expressing, RNA Immunoprecipitation, Modification, Binding Assay, Real-time Polymerase Chain Reaction, Over Expression, Knockdown, Standard Deviation

Journal: Nature Communications

Article Title: Deep learning-assisted discovery of a potent and cell-active inhibitor of RNA N 6 -methyladenosine recognition protein YTHDC2

doi: 10.1038/s41467-025-65542-0

Figure Lengend Snippet: a Western blot analyses of H3K4me3 expression in DC2-C1 -treated PANC-1 or MiaPACA-2 cells. Blots shown are representative of three biological replicates. Full blots can be found in Supplementary Fig. . b Quantitative analysis of Western blot for PANC-1 or MiaPACA-2 cells. Data are shown as the mean ± SD from three technical replicates in three independent experiments. c Colony formation assay of PANC-1 or MiaPACA-2 cells treated with DC2-C1 or 16o . d Abilities of invasion of PANC-1 or MiaPACA-2 cells treated with DC2-C1 or 16o . Scale bar, 50 μm. e-g ACC1 , FASN and SREBP-1C mRNA expression were determined by RT-qPCR in DC2-C1 -treated HepG2 cells. Data are shown as the mean ± SD from three technical replicates in three independent experiments. h Western blot analyses of ID2 expression in DC2-C1 -treated PANC-1 cells; Western blot analyses of FZD7 expression in DC2-C1 -treated HGC27 cells. Blots shown are representative of three biological replicates. Full blots can be found in Supplementary Fig. . i FZD7 mRNA expression was determined by RT-qPCR in DC2-C1 -treated HGC27 cells. Data are shown as the mean ± SD from four technical replicates in four independent experiments. j ID2 mRNA expression was validated by RT-qPCR in DC2-C1 -treated PANC-1 cells. Data are shown as the mean ± SD from four technical replicates in four independent experiments. Source data are provided as a Source Data file. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; by two-tailed unpaired Student's t-test).

Article Snippet: Antibodies used: YTHDC2 antibody (ZenBio, R27443 ), AMIGO2 antibody (ImmunoWay, YN2372), ID2 antibody (HUABIO, M1301-2), H3K4me3 antibody (Abcam, ab8580), FZD7 antibody (Proteintech, 16974-1-AP), DYKDDDDK Tag (Flag-tag) antibody (Cell Signaling Technology, 14793), GAPDH antibody (Proteintech, 60004-1-Ig), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00001-2), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00001-1).

Techniques: Western Blot, Expressing, Colony Assay, Quantitative RT-PCR, Two Tailed Test