Fig. 5: YTHDF3 promotes the translation of INCENP and CDCA8 mRNAs.

a, b Representative images and analysis of INCENP and CDCA8 protein expression levels after YTHDF3 knockdown in PDX-derived organoids (PDXO) by immunofluorescence. Scale bar = 10 μm. Mean protein fluorescence intensity was determined using ImageJ software. c, d Gene plots illustrating YTHDF3 binding to INCENP and CDCA8 mRNAs, as measured by RIP-seq. The normalized read distribution: input (yellow) and YTHDF3 (purple) along the mRNAs. e RNA immunoprecipitation-qPCR (RIP-qPCR) highlighting the indicated mRNA transcripts bound by YTHDF3 in ESCC cells. f, g Representative immunofluorescence staining of newly synthesized proteins and statistical analysis in KYSE30 and KYSE150 cells after YTHDF3 overexpression. Relative fluorescence intensity was determined using ImageJ software. Puromycin staining (green) indicates newly synthesized proteins; nuclei are stained with DAPI (blue). Scale bar = 50 μm. h, i Mock or YTHDF3 knockdown KYSE30 and KYSE150 cells were transfected with pmirGLO-INCENP or pmirGLO-CDCA8 reporters for 48 h. Translation efficiency of INCENP or CDCA8 is defined as the quotient of reporter protein production (F-luc/R-luc). j, k Co-immunoprecipitation of endogenous YTHDF3 and eIF3A in ESCC cells. Representative immunoblots shown in figures were repeated three times independently with similar results. l, m In situ detection and quantification of YTHDF3–eIF3A interactions in the indicated ESCC cell lines were measured using PLA assays, and puncta per cell were determined using ImageJ software. Scale bar = 10 μm, n = 5 biological replicates. n RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in YTHDF3 knockdown KYSE30 cells. o RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in Ebselen-treated KYSE30 cells. RIP-qPCR illustrating the association of YTHDF3 (p) and eIF3A (q) with the indicated transcripts in METTL3 knockdown KYSE30 cells. Data were analyzed by two-tailed unpaired t-test (b, e, g, h, i, m) or one-way ANOVA (n, o, p, q). n = 3 biological replicates (b, e, g, h, i, n, o, p, q). Data were presented as means ± SD. Source data are provided as a Source Data file.