Fig. 6

HNRNPC binds to the ALKBH5-mediated m6A modification of DDX58 mRNA. a The flowchart of ChIRP assay is shown using biotin-labeled DDX58 mRNA probes. b The bubble chart shows the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the immunoprecipitated proteins from ChIRP coupled with mass spectrometry. c The top 20 precipitated proteins are shown in the table. d The identification of HNRNPC by mass spectrometry is shown. e The immunoprecipitated proteins from ChIRP were detected using immunoblotting with an anti-HNRNPC antibody. f RNA binding protein immunoprecipitation (RIP) assays were performed using an HNRNPC antibody or IgG after si-ALKBH5 transfection for 48 h. Two primers targeting the m6A-modified region of DDX58 mRNA were used in the RIP-PCR. g, h Pre-mRNA and mature DDX58 mRNA were analyzed using qPCR after the ectopic expression of HNRNPC or ALKBH5 silencing for 48 h. i RIG-I was detected by immunoblotting assays after transfection with HNRNPC expression plasmids for 48 h. j RIG-I expression was detected after cotransfection with HNRNPC expression plasmids and siRNA for RIG-I. k RIG-I was detected after transfection with siRNA for HNRNPC for 48 h. l RIG-I expression was detected after cotransfection with HNRNPC- and RIG-I-siRNA expressing plasmids for 48 h. m, n The correlations between HNRNPC and DDX58 mRNA or RIG-I protein were analyzed in the HNSCC tissue microarray and TCGA dataset. o, p Luciferase vectors with the wild-type (WT) or m6A nucleotide-mutated (MT) DDX58 gene were transfected into 293 T cells with HNRNPC overexpression or depletion. Relative luciferase activity was measured. Data information: Data are presented as the mean ± S.D. Two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01