Fig. 6

DHX58 recruits YTHDC2 to promote the translation of m⁶A-modified Gpx4 mRNA. a The endogenous association between DHX58 and YTHDC2 in liver tissues was examined by Co-IP. b GPX4 protein level in primary hepatocytes with Ythdc2 overexpression or knockdown was examined by Western blotting. c In primary hepatocytes with Ythdc2 overexpression or knockdown, the relative distribution of Gpx4 mRNA in each ribosome fraction was analyzed by qRT-PCR. ^##P < 0.01 vs. Empty vector. d In primary hepatocytes from Dhx58^f/f and Dhx58^hep−/− mice with knockdown of Ythdc2, DHX58, YTHDC2, and GPX4 protein levels were examined by Western blotting. e Sequencing read clusters from MeRIP-seq analysis of Gpx4 mRNA in primary hepatocytes and top consensus motif identified by HOMER with MeRIP-seq peaks. f m⁶A modification of Gpx4 and Acsl4 mRNAs in primary hepatocytes were examined by RIP-qRT-PCR. g GPX4 protein level in primary hepatocytes with knockdown of Mettl3 was examined by Western blotting. h In primary hepatocytes transfected with Flag-tagged DHX58 or YTHDC2, together with Mettl3 knockdown, Flag-tag, METTL3, and GPX4 protein levels were examined by Western blotting. Data are shown as mean ± SD (n = 3) or photographs from one representative of three independent experiments. ^**P < 0.01. ns non-significant, Si-1 No.1 siRNA targeting YTHDC2, Si-2 No.2 siRNA targeting YTHDC2, DHX58 DExH-box helicase 58, YTHDC2 YT521-B homology domain containing 2, m⁶A N⁶-methyladenosine, Gpx4 glutathione peroxidase 4, Co-IP co-immunoprecipitation, MeRIP-seq methylated RNA immunoprecipitation sequencing, HOMER hypergeometric optimization of motif enrichment, Acsl4 acyl-CoA synthetase long chain family member 4, RIP RNA immunoprecipitation, METTL3 methyltransferase complex methyltransferase-like 3, CTD C-terminal domain, SD standard deviation, LMW low molecular weight, HMW high molecular weight