Fig. 4

YTHDF2 processes m⁶A-marked IL11 and SERPINE2 mRNAs for decay. (a) m⁶A-enrichment in the IL11 and SERPINE2 mRNAs in SMMC7721 cells expressing empty vector (EV) or overexpressing YTHDF2 (OE), as assessed by MeRIP-qPCR. The cells were subjected to Nx or Hx for 12 h. n = 3 biological replicates. (b) Gene enrichment of IL11 and SERPINE2 in YTHDF2-mRNA complex immunoprecipitated from SMMC7721-OE cells, as determined by RIP-qPCR. The cells were subjected to Nx or Hx for 12 h. n = 3 biological replicates. (c, d) RNA lifetime of IL11 and SERPINE2 in indicated SMMC7721 cells, as determined by monitoring transcript abundance after transcription inhibition (TI). The cells were subjected to Nx or Hx 8 h prior to TI initiation. n = 3 biological replicates. (e) The average read density showing the m⁶A peaks identified in IL11 and SERPINE2 transcripts in human HCC tissues as assessed using MeRIP-seq. n = 8 patients. (f) Luciferase reporter assay showing posttranscriptional regulation by YTHDF2 in the presence of IL11 (left) or SERPINE2 (right) 3’UTR. Indicated SMMC7721 cells were grown for 12 h under Nx or Hx. Renilla luciferase activity was normalized to firefly activity and presented as relative luciferase activity. n = 3 biological replicates. (g) Immunofluorescence staining of YTHDF2 and DCP1a (P-body marker) in YTHDF2-overexpressing SMMC7721 cells grown for 12 h under Nx or Hx. n = 3 biological replicates. Scale bar, 5 μm. (h) Fluorescence in situ hybridization of IL11 or SERPINE2 mRNA and immunofluorescence staining of DCP1a in indicated SMMC7721 cells grown for 12 h under Nx or Hx. n = 3 biological replicates. Scale bar, 5 μm. Error bars indicate means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. P-values were determined by two tailed t-test