Journal: Nature communications

Article Title: Adipocyte YTH N(6)-methyladenosine RNA-binding protein 1 protects against obesity by promoting white adipose tissue beiging in male mice.

doi: 10.1038/s41467-023-37100-z

Figure Lengend Snippet: Fig. 1 | Adipose YTHDF1 expression is reduced in obesity. a, b Immunoblot analysis of YTH family proteins in iWAT a and BAT b of mice fed with CD or HFD for 12 weeks. c, d mRNA levels of Yth family genes and thermogenesis-related genes in iWAT c and BAT d of CD- and HFD-fed mice. Data were presented as mean ± SEM

Article Snippet: Conditional Ythdf1 KO allele (Ythdf1CTL) was generated by Cyagen Biosciences (China).

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: m 6 A Methyltransferase KIAA1429 Regulates the Cisplatin Sensitivity of Gastric Cancer Cells via Stabilizing FOXM1 mRNA.

doi: 10.3390/cancers14205025

Figure Lengend Snippet: Figure 5. Effect of YTHDF1 on regulating FOXM1 expression. (A) RNA immunoprecipitation (RIP) indicated the direct binding within YTHDF1 and FOXM1 mRNA in AGS Cis-R or NCI-N87 Cis-R and their parental wild type cells. (B,C) The mRNA level of FOXM1 in YTHDF1 shRNA infected (B) or YTHDF1 overexpressed (C) AGS Cis-R or NCI-N87 Cis-R cells was assessed by QRT-PCR. (D) RNA immunoprecipitation (RIP) indicated the direct binding within YTHDF1 and FOXM1 mRNA in KIAA1429 shRNA infected AGS Cis-R or NCI-N87 Cis-R cells. (E,F) RNA decay rate assay demonstrated the FOXM1 mRNA half-lives upon the YTHDF1 knockdown in AGS Cis-R (E) or NCI- N87 Cis-R (F) cells. Data were detected at indicated timepoint with actinomycin D (Act D, 5 µg/mL) treatment. (G) Correlation analysis by Spearman’s rank correlation coefficient (GEPIA) showed the correlation between YTHDF1 and FOXM1 in the gastric cancer tissue specimens. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Plasmid for p65 overexpression was obtained from addgene [31]; pCMV6-Entry, KIAA1429, and YTHDF1 overexpression plasmids were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, RNA Immunoprecipitation, Binding Assay, shRNA, Infection, Quantitative RT-PCR, Knockdown

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: 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: 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: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Primer Sequences used for qRT-PCR.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques:

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: m 6 A reader protein YTHDF1 promotes autophagy activation and BECN1 mRNA stability via recognizing the m 6 A binding site. HSC-T6 and HSC-LX2 cells were added sorafenib (10 μM), erastin (10 μM), and RSL3 (2.5 μM) and treated for 24 h. ( A ) Western blot was used to assay the protein levels of m 6 A readers (n = 3 in every group). ( B ) The mRNA levels of m 6 A readers were determined by real-time PCR (***, p < 0.001, n = 3 in every group). ( C ) YTHDF1 plasmid or control vector were transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h, and then were pretreated with Act-D (5 μg/ml) for indicated times. Real-time PCR was used to measure the remaining BECN1 mRNA levels (*, p < 0.05, n = 3 in every group). ( D ) YTHDF1 plasmid or control vector were transfected into HSC-LX2 and treated with erastin (10 μM) for 24 h. Western blot was used to determine the protein levels of BECN1 at different times under the CHX (100 μg/ml) treatment (n = 3 in every group). ( E ) The binding of YTHDF1 and BECN1 mRNA was measured by ribonucleoprotein immunoprecipitation (RNP IP) (**, p < 0.01, n = 3 in every group). ( F ) biotinylated transcripts of the BECN1 mRNA 3′-UTR, CDS, 5′-UTR were used for mRNA affinity isolation assay (n = 3 in every group). ( G ) HSC-LX2 cells were transfected with control vector or YTHDF1 plasmid and by erastin (10 μM) treatment for 24 h. The levels of m 6 A modification in BECN1 mRNA 3′-UTR, CDS and 5′-UTR were determined by MeRIP qPCR (**, p < 0.01, n = 3 in every group). ( H ) HSC-LX2 cells were stably transfected with empty vector (pGL3)or luciferase constructs carrying the genes encoding 5′-UTR-BECN1, CDS-BECN1, 3′-UTR-BECN1. Then cells were added erastin (10 μM) and treated for 24 h, and luciferase activities were measured (**, p < 0.01, n = 3 in every group). ( I ) BECN1 mRNA m 6 A binding site was showed. ( J ) Schematic representation of positions of m 6 A motifs and mutation in CDS within BECN1 mRNA were showed. ( K ) Binding of YTHDF1 with the BECN1-CDS-WT, BECN1-CDS-Mut1 (A437G) and BECN1-CDS-Mut2 (A1276G) in HSC-LX2 were analyzed by YTHDF1 RNP IP (**, p < 0.01, n = 3 in every group). ( L ) HSC-LX2 cells were transfected with control vector or YTHDF1 plasmid and BECN1-CDS-WT, BECN1-CDS-Mut1 and BECN1-CDS-Mut2 plasmid, and then were pretreated with Act-D (5 μg/ml) for indicated times. Real-time PCR was used to measure the remaining BECN1 mRNA (*, p < 0.05, n = 3 in every group). ( M ) After transfected with BECN1-CDS-WT, BECN1-CDS-Mut1 and BECN1-CDS-Mut2 plasmid, HSC-LX2 cells were treated with erastin (10 μM) for 48 h. The protein expression of BECN1 was measured by western blot (*, p < 0.05, ***, p < 0.001, n = 3 in every group).

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Activation Assay, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Transfection, Immunoprecipitation, Isolation, Modification, Stable Transfection, Luciferase, Construct, Mutagenesis, Expressing

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: HSC-specific inhibition of m 6 A modification impairs erastin-induced HSC ferroptosis in murine liver fibrosis. Mice of 6 groups were treated with Vehicle, CCl 4, CCl 4 +VA-Lip-control-vector + Erastin, CCl 4 +VA-Lip-Mettl4-shRNA + Erastin, CCl 4 +VA-Lip-Fto-Plasmid + Erastin, CCl 4 +VA-Lip-Ythdf1-shRNA + Erastin. ( A ) Macroscopic examination was used to observe the pathological changes of the livers. Scale bars: 1 cm. Histopathological study was performed by H&E, Masson, and Sirius Red staining. Representative photographs were showed. Scale bars: 50 μm. (***, p < 0.001, n = 6 in every group). ( B ) Immunohistochemical staining of α-SMA was determined. Representative photographs were showed. Scale bars: 50 μm.(***, p < 0.001, n = 6 in every group). ( C ) Real-time PCR was measured to determine the mRNA expression of liver fibrosis markers (Acta2, Col1a1, Fn1, and Des) (*, p < 0.05, **, p < 0.01, ***, p < 0.001, n = 6 in every group). ( D ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (n = 6 in every group, ***, p < 0.001, N.S., not significant). ( E, F ) Real-time PCR was used to determine the mRNA expression of autophagy markers (Becn1, Map1lc3b, Sqstm1, and Fth1) (*, p < 0.05, ***, p < 0.001, N.S., not significant, n = 6 in every group). ( G ) The mRNA expression of Ptgs2, iron accumulation and MDA production were determined (*, p < 0.05, **, p < 0.01, ***, p < 0.001, N.S., not significant, n = 6 in every group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Inhibition, Modification, Control, Plasmid Preparation, shRNA, Staining, Immunohistochemical staining, Real-time Polymerase Chain Reaction, Expressing, Methylation

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: m 6 A modification upregulation, autophagy activation, and ferroptosis induction occur in human HSCs receiving sorafenib monotherapy. ( A, B ) laser capture microdissection (LCM) was used to isolated the primary human HSCs from the collected liver tissue. ACTA2, FN1, COL1A1, METTL4, FTO, and YTHDF1 mRNA expression were determined by real-time PCR (No treatment, n = 10; Sorafenib treatment, n = 10, **, p < 0.01, ***, p < 0.001). ( C ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( D ) Real-time PCR was used to determine the mRNA expression of autophagy markers BECN1, MAP1LC3B, SQSTM1, and FTH1 (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( E ) The PTGS2 mRNA expression, iron accumulation, MDA production and GSH depletion were determined (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001).

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Activation Assay, Laser Capture Microdissection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Methylation

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: m 6 A modification induces HSC ferroptosis by regulating autophagy signaling pathway. The upregulation of methylase METTL4 and the downregulation of demethylase FTO increased the levels of m 6 A modifications in BECN1 mRNA. m 6 A reader YTHDF1 promoted BECN1 mRNA stability via recognizing the m 6 A binding site, thus triggering autophagy activation, and eventually leading to HSC ferroptosis.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Binding Assay, Activation Assay