K Lysates from transfected HEK 293 T cells with Myc-tagged USP7 (WT) or Myc-tagged USP29 (C223S), together with HA-tagged Ub and Flag-tagged FTO, were generated after immunoprecipitation with anti-Flag and immunoblotting with anti-HA and anti-Flag. L The Myc-USP7, Flag-FTO and the...
siRNAs targeting FTO, ACSL4 and TFRC were generated by GenePharma (Shanghai, China). The transfection of plasmids was performed using ViaFect Transfection Reagent (E4982, Promega, Madison, USA) according to the manufacturer’s instructions. Transient knockdown of target genes by siRNA was performed...
Since m6A modifications are generated by methyltransferases and removed by demethyltransferases in eukaryotic RNA, we systematically analysed the mRNA levels of m6A modification enzymes in TC and Nor tissues (Fig. 1D; Supplement Fig. 1A-E). The mRNA levels of the demethyltransferases FTO and ALKB...
The FTO transgenic mice were generated by microinjecting the transgenic construct into a fertilized egg using CRISPR/Cas9-mediated genome editing. In brief, the FTO knock-in (FTOKI) sgRNA were constructed based on Fto gene (NM_011936). CRISPR/Cas9 components, including Cas9, donor vector, and...
Finally, we generated a conformational ensemble for apo FTO that satisfies the solution NMR data by combining the experimental residual dipolar couplings with accelerated molecular dynamics simulations. Altogether, the structural ensemble reported in this work provides an atomic-resolution model of apo FTO...
Accession code: Miccroarray data generated by hybridization of RNA from both FTO1_C1 and FTO2_D4 clones with and without induced expression as well as from FTO siRNA- and scrampled siRNA-treansfected cells can be obtained from Gene Expression Omnibus (GSE33870). URL: GeneTrail gene set ...
I Mutations in m6A consensus sequences were generated by replacing adenosine with thymine. J Relative luciferase activity of the wild-type and 3-mutant GEF-H1 3'UTR reporter vectors in FTO-overexpressing HESCs. *P < 0.05, **P < 0.005, ***P < 0.001. K GEF-H1 expression was...
The experimental data were representative of three independent experiments at least, and the GraphPad Prism software (version 9.0 for Windows) was used for calculations and generated the statistical graph. The differences between two groups were analyzed using two-tailed t-tests. One-way analysis of...
(Fig.4e). The m6A modification is a dynamically reversible process, added by methyltransferases (Writers: METTL3, METTL14) and removed by demethylases (Erasers: FTO, ALKBH5) [15]. Here, we generated METTL3 and METTL14 knockdown cells with the decreased total m6A levels (Fig.4f) and found...
Further information and requests for resources and reagents may be directed to and will be fulfilled by the Lead Contact, Jianjun Chen (jianchen@coh.org). Materials Availability All cell lines, plasmids, and other stable reagents generated in this study are available from the Lead Contact with ...