Figure 5.

YTHDF1 regulates TFRC expression in HPSCC cells in an m6A methyltransferase-dependent manner. (A) Relative RNA level of TFRC in Detroit 562 and FaDu cells upon YTHDF1 knockdown. (B) Western blot analysis of the protein level of TFRC in Detroit 562 and FaDu cells upon YTHDF1 knockdown. (C,D) RIP analysis of the interaction of the 5'UTR (C) and 3'UTR (D) of TFRC mRNA in FaDu cells transfected with the FLAG-YTHDF1-WT plasmid. Enrichment of TFRC with FLAG was measured by qPCR and normalized to the input level. (E) Western blot analysis of the protein level of TFRC in Detroit 562 and FaDu cells transfected with the YTHDF1-WT or YTHDF1-Mut plasmid. (F) Schematic representation of wild-type (TFRC-WT) and m6A mutant (TFRC-Mut) TFRC constructs. (G) Relative luciferase activity of the WT or Mut TFRC-5′UTR and TFRC-3′UTR luciferase reporter in FaDu cells transfected with control vector or shYTHDF1. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (H) Relative luciferase activity of WT and Mut (A-to-T mutation) TFRC-5′UTR and TFRC- 3′UTR luciferase reporters in Detroit 562 cells transfected with pCMV-YTHDF1-WT or pCMV-YTHDF1-Mut plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (I,J) The m⁶A modification in the 5'UTR (I) and 3'UTR (J) of TRFC mRNA in Detroit 562 and FaDu cells with YTHDF1 knockdown, as assessed by gene-specific m⁶A-RIP-qPCR assays. Error bars indicate the means ± SEM, n = 3; unpaired Student's t-test.