Fig. 4

WTAP repressed ETS1 in an m6A-HuR mediated pattern. a The m6A level of poly(A) + RNAs isolated from total RNA of WTAP-knockdown Huh7 and PLC/PRF/5 cells was indicated by m6A dot blot. Corresponding RNAs were loaded equally by a 2-fold serial dilution with 400 ng, 200 ng and 100 ng. Methylene blue staining served as a loading control; b The global content of m6A was also examined by RNA methylation quantification assay, relying on the standard curve; c MeRIP analysis followed by qRT-PCR was applied to assess the m6A modification of ETS1 in two WTAP-silencing HCC cells. The enrichment of m6A in each group was calculated by m6A-IP/input and IgG-IP/input. d Three luciferase plasmids were constructed by inserting the corresponding cDNAs into pGL3-control vectors. Wild-type reporters embodied the full-length 3’UTR and a partial CDS sequence near stop codon of ETS1 with intact m6A sites, while mutant ones obtained some A-C mutations on m6A consensus motifs (Mut1 or Mut2 contained 11 or 4 mutations, respectively). Luciferase activity was detected and normalized to Renilla activity; e Relative activity of the WT or Mut luciferase reporters in WTAP-silenced Huh7 and PLC/PRF/5 cells was determined (normalized to negative control groups); f ETS1 expression was identified by western blotting in Hep3B and SMMC-7721 cells upon knockdown of HuR (#1, #2) compared with siNC; g Immunoprecipitation of HuR-related RNA in control or WTAP-knockdown cells was conducted followed by RT-qPCR to detect the amount of ETS1 mRNA binding to HuR; h ETS1 expression was measured by RT-qPCR in Huh7 and MHCC97H cells with or without knockdown of WTAP or HuR compared with NC; i The RNA decay rate was determined in Huh7 and MHCC97H cells after treatment with Actinomycin D (normalized to 0 h); j The relative activity of the WT or Mut luciferase reporters was detected in WTAP/HuR-rescued Huh7 and MHCC97H cells (normalized to negative control groups)