Journal: Journal of Translational Medicine

Article Title: Stage-specific requirement for METTL3-dependent m 6 A modification during dental pulp stem cell differentiation

doi: 10.1186/s12967-022-03814-9

Figure Lengend Snippet: The dynamic m 6 A epitranscriptomic landscape in DPSC differentiation. A DPSCs were induced with osteo/odontogenic medium for 0, 7, and 14 days, and then the total m 6 A level in the RNA polls was quantified by the colorimetric method. B The m 6 A methylation landscape in the genome-wide transcriptome was evaluated by m 6 A RIP-seq. Distribution of m 6 A peaks in the 3′ UTR, CDS region and 5′ UTR. C Pie chart analysis of the m 6 A peak fraction in transcript segments. D Specific and conserved motif sequences of high-confidence m 6 A peaks identified by the HOMER database. E Epitranscriptome profile of m 6 A-tagged mRNAs in DPSCs after odontogenic differentiation. F Gene Ontology enrichment analysis of differentially m 6 A methylated and expressed transcripts. G The concentrations of SAM, SAH and potential of m 6 A establishment were qualified by LC‒MS/MS. Metabolites with a VIP value > 1 were further subjected to statistical analysis (n = 6). H The mRNA expression of METTL3, METTL14, and WTAP during DPSC mineralization, as evaluated by qPCR (n = 3). I The protein expression of METTL3, METTL14, and WTAP in DPSCs was evaluated by western blotting. SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine. Significance was determined via ANOVA or Student’s t test. The quantitative data are presented as the mean ± SD * p < 0.05. ** p < 0.01. *** p < 0.001

Article Snippet: Primary and secondary antibodies against the following proteins were used in this study: METTL3 (96391, 1:1000); METTL14 (51104, 1:1000); WTAP (56501, 1:1000); p-Smad1/5 (9516, 1:1000) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Methylation, Genome Wide, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Unraveling the molecular complexity: Wtap/Ythdf1 and Lcn2 in novel traumatic brain injury secondary injury mechanisms

doi: 10.1007/s10565-024-09909-x

Figure Lengend Snippet: Wtap/Ythdf1 Mediates Lcn2 m6A Modification. ( A ) Western blot analysis of protein expression of Wtap, Ythdf1, and Lcn2 in primary cortical neurons of different groups; ( B - C ) Quantification of m6A RNA methylation and m6A dot plot experiments to detect m6A modification levels in primary cortical neurons of different groups; ( D ) RT-qPCR analysis of mRNA expression of Lcn2 in primary cortical neurons of different groups; ( E ) MeRIP qPCR analysis of m6A modification levels of Lcn2 in primary cortical neurons of different groups; ( F ) RIP PCR analysis of the enrichment of Wtap and Ythdf1 on Lcn2 mRNA; ( G - H ) RT-qPCR and Western blot analysis of Lcn2 expression in primary cortical neurons after Wtap or Ythdf1 knockout; ( I ) Quantification of m6A RNA methylation and m6A dot plot experiments to detect changes in m6A modification levels after Wtap or Ythdf1 knockout; ( J ) MeRIP qPCR analysis of m6A modification levels of Lcn2 after Wtap or Ythdf1 knockout; ( K ) RIP PCR analysis of the enrichment of Ythdf1 on Lcn2 mRNA after Wtap knockout; ( L ) RT-qPCR analysis of the mRNA half-life of Lcn2 after Wtap or Ythdf1 knockout, Act D (actinomycin D, 5 µg/mL); ( M ) Schematic representation of the location and mutation of m6A motifs in the coding sequence (CDS) of Lcn2 mRNA; ( N ) RIP PCR analysis of the binding of m6A, Wtap, and Ythdf1 to Lcn2 mRNA before and after Lcn2 mutation. * indicates P < 0.05 compared to the Control, vector, or Lcn2-WT group; cell experiments repeated 3 times

Article Snippet: The PVDF membrane was then incubated in 5% skim milk powder and sealed at room temperature for 1 h. Primary antibodies, including anti-Wtap antibody (mouse, 1:5000, 60188–1-Ig, Proteintech, Wuhan, China) or anti-Ythdf1 antibody (rabbit, 1:1000, 17479–1-AP, Proteintech, Wuhan, China), anti-Lcn2 antibody (rabbit, 1:500, A2092, Abclonal, Wuhan, China), anti-cleaved caspase-3 antibody (rabbit, 1:1000, #9661, Cell Signaling Technology, USA), anti-Bax antibody (rabbit, 1:1000, #2772, Cell Signaling Technology, USA), anti-Bcl-2 antibody (rabbit, 1:1000, #3498, Cell Signaling Technology, USA), and rabbit anti-β-actin (1:1000, ab8226, Abcam, UK) were introduced and allowed to incubate overnight at 4 °C.

Techniques: Modification, Western Blot, Expressing, Methylation, Quantitative RT-PCR, Knock-Out, Mutagenesis, Sequencing, Binding Assay, Control, Plasmid Preparation

Journal: Cell Biology and Toxicology

Article Title: Unraveling the molecular complexity: Wtap/Ythdf1 and Lcn2 in novel traumatic brain injury secondary injury mechanisms

doi: 10.1007/s10565-024-09909-x

Figure Lengend Snippet: Regulation of Lcn2 Expression by Wtap/Ythdf1 Affects Neuronal Death in TBI Model. ( A - B ) RT-qPCR and Western blot analysis of Lcn2 expression in primary cortical neurons of different groups; ( C ) Detection of LDH release in primary cortical neurons of different groups; ( D ) Flow cytometry analysis of apoptosis in primary cortical neurons of different groups; ( E ) Western blot analysis of the expression of apoptosis-related proteins in primary cortical neurons of different groups; ( F ) ELISA analysis of the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in primary cortical neurons of different groups. * indicates P < 0.05 compared to the vector + oe-NC group, # indicates P < 0.05 compared to the Wtap-KO + oe-NC group, & indicates P < 0.05 compared to the Ythdf1-KO + oe-NC group; cell experiments repeated 3 times

Article Snippet: The PVDF membrane was then incubated in 5% skim milk powder and sealed at room temperature for 1 h. Primary antibodies, including anti-Wtap antibody (mouse, 1:5000, 60188–1-Ig, Proteintech, Wuhan, China) or anti-Ythdf1 antibody (rabbit, 1:1000, 17479–1-AP, Proteintech, Wuhan, China), anti-Lcn2 antibody (rabbit, 1:500, A2092, Abclonal, Wuhan, China), anti-cleaved caspase-3 antibody (rabbit, 1:1000, #9661, Cell Signaling Technology, USA), anti-Bax antibody (rabbit, 1:1000, #2772, Cell Signaling Technology, USA), anti-Bcl-2 antibody (rabbit, 1:1000, #3498, Cell Signaling Technology, USA), and rabbit anti-β-actin (1:1000, ab8226, Abcam, UK) were introduced and allowed to incubate overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Cell Biology and Toxicology

Article Title: Unraveling the molecular complexity: Wtap/Ythdf1 and Lcn2 in novel traumatic brain injury secondary injury mechanisms

doi: 10.1007/s10565-024-09909-x

Figure Lengend Snippet: Regulation of brain tissue and neurofunctional disorders in TBI mice by the Wtap/Lcn2 axis. ( A - B ) Expression of Wtap, Ythdf1, and Lcn2 in the cortical tissues of mice from each group detected by RT-qPCR and Western blot; (C-E) mNSS scores ( C ), foot fault rates ( D ), and latency to fall in the rotarod test ( E ) of mice from each group; ( F ) Escape latency and time to cross the platform in the MWM test of mice from each group; ( G ) Contusion volume in the ipsilateral cortex of mice from each group (scale bar = 1 mm); ( H ) Water content in the brain tissues of mice from each group. * indicates difference from Sham + sh-NC + oe-NC group, p < 0.05, # indicates difference from TBI + sh-NC + oe-NC group, p < 0.05, & indicates difference from TBI + sh-Wtap + oe-NC group, p < 0.05, each group consisted of 8 mice

Article Snippet: The PVDF membrane was then incubated in 5% skim milk powder and sealed at room temperature for 1 h. Primary antibodies, including anti-Wtap antibody (mouse, 1:5000, 60188–1-Ig, Proteintech, Wuhan, China) or anti-Ythdf1 antibody (rabbit, 1:1000, 17479–1-AP, Proteintech, Wuhan, China), anti-Lcn2 antibody (rabbit, 1:500, A2092, Abclonal, Wuhan, China), anti-cleaved caspase-3 antibody (rabbit, 1:1000, #9661, Cell Signaling Technology, USA), anti-Bax antibody (rabbit, 1:1000, #2772, Cell Signaling Technology, USA), anti-Bcl-2 antibody (rabbit, 1:1000, #3498, Cell Signaling Technology, USA), and rabbit anti-β-actin (1:1000, ab8226, Abcam, UK) were introduced and allowed to incubate overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Unraveling the molecular complexity: Wtap/Ythdf1 and Lcn2 in novel traumatic brain injury secondary injury mechanisms

doi: 10.1007/s10565-024-09909-x

Figure Lengend Snippet: Schematic representation of the molecular mechanism by which the Wtap/Ythdf1-mediated neural Lcn2 m6A modification regulates the activation of astrocytes and microglia, affecting secondary damage in TBI mice

Article Snippet: The PVDF membrane was then incubated in 5% skim milk powder and sealed at room temperature for 1 h. Primary antibodies, including anti-Wtap antibody (mouse, 1:5000, 60188–1-Ig, Proteintech, Wuhan, China) or anti-Ythdf1 antibody (rabbit, 1:1000, 17479–1-AP, Proteintech, Wuhan, China), anti-Lcn2 antibody (rabbit, 1:500, A2092, Abclonal, Wuhan, China), anti-cleaved caspase-3 antibody (rabbit, 1:1000, #9661, Cell Signaling Technology, USA), anti-Bax antibody (rabbit, 1:1000, #2772, Cell Signaling Technology, USA), anti-Bcl-2 antibody (rabbit, 1:1000, #3498, Cell Signaling Technology, USA), and rabbit anti-β-actin (1:1000, ab8226, Abcam, UK) were introduced and allowed to incubate overnight at 4 °C.

Techniques: Modification, Activation Assay

Journal: eLife

Article Title: A cleaved METTL3 potentiates the METTL3–WTAP interaction and breast cancer progression

doi: 10.7554/eLife.87283

Figure Lengend Snippet: ( a ) Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T transfected with METTL3 containing different tag. ( b ) Immunoblot analysis showing the binding between purified METTL3-Flag and GST-METTL3. ( c ) Immunoblot of WCE and IP of 293T transfected with METTL3 containing different tag with or without RNase or DNase. ( d ) A schematic representation of METTL3 and its truncations used for IP. ( e ) Immunoblot of WCE and IP of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with full-length (FL) or truncation of METTL3. ( f ) Immunoblot of WCE and IP of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with METTL3-WT-flag, METTL3-Δ238-Flag combining with or without METTL3a (no flag tag) or control vector as indicated. Immunoblot of WCE and IP of 293T cells infected with or without lentivirus encoding METLL3 shRNA ( g ) or METTL14 shRNA ( h ) followed by transfection with METTL3 or METTL14 as indicated. ( i ) Immunoblot of WCE and IP of 293T cells infected with lentivirus encoding METTL3 shRNA followed by transfection with WTAP containing different tag. ( j, k ) Immunoblot of WCE and IP of 293T cells infected with lentivirus encoding WTAP shRNA followed by transfection with METTL3 or METTL14 as indicated. N 6 -methyladenosine (m 6 A) dot blot ( l ) and quantification ( m ) of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with METTL3-WT, METTL3 variants, or control vector as indicated. Methylene blue (MB) is used as a loading control. m 6 A dot blot ( n ) and quantification ( o ) of 293T cells with WT or CRISPR knock-in mediated deletion of Q238 with or without transfection of METTL3a. FL indicates the full-length of METTL3. The short forms are labeled as a and b. no tag represents exogenous METTL3a containing no flag tag. Figure 5—source data 1. Unedited western blot images for . Figure 5—source data 2. Tables related to .

Article Snippet: Antibodies against METTL14 (26158-1-AP), WTAP (mouse, 60188-1-Ig; rabbit, 10200-1-AP), PSMC3 (24142-1-AP), PSMD10 (12342-2-AP), mTOR (66888-1-Ig), TMEM127 (23142-1-AP), Raptor (20984-1-AP), Rictor (27248-1-AP), TSC1 (29906-1-AP), TSC2 (24601-1-AP), LKB1 (10746-1-AP), PIK3CA (27921-1-AP), and rabbit Flag (20543-1-AP) were obtained from Proteintech.

Techniques: Western Blot, Transfection, Binding Assay, Purification, Infection, shRNA, FLAG-tag, Control, Plasmid Preparation, Dot Blot, CRISPR, Knock-In, Labeling