Fig 2: YTHDF1 promotes ACSL3 translation in an m6A-dependent manner. (A,B) ACSL3 expression detected by RT-qPCR and Western blot. (C,D) Polysome profiling analysis. (E) RIP assay. (F,G) ACSL3 expression detected by Western blot. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. RIP, RNA immunoprecipitation; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation.

Fig 3: Upregulation of ACSL3 attenuates the inhibitory effects of YTHDF1 downregulation on proliferation in READ cells. (A) ACSL3 expression detected by Western blot. (B) Cell viability measured by CCK-8 assay. (C) Cell proliferation assessed by colony formation assay. (D,E) Cell cycle and apoptosis analyzed by flow cytometry. Data are presented as mean ± SD. *, P<0.05; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; PI, propidium iodide; READ, rectal adenocarcinoma; SD, standard deviation.

Fig 4: Downregulation of ACSL3 suppresses proliferation in READ cells. (A,B) Validation of ACSL3 knockdown and overexpression by RT-qPCR and Western blot. (C) Cell viability measured by CCK-8 assay. (D) Cell proliferation assessed by colony formation assay. (E,F) Cell cycle and apoptosis analyzed by flow cytometry. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; PI, propidium iodide; READ, rectal adenocarcinoma; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation.

Fig 5: ACSL3 is an m6A-modified target gene of YTHDF1. (A) ACSL3 expression in the TCGA-READ dataset. (B) Correlation between YTHDF1 and ACSL3 expression in the TCGA-READ dataset. (C) m6A modification sites on ACSL3 mRNA predicted by RMBase v3.0. (D) YTHDF1 binding sites on ACSL3 mRNA predicted by ENCORI. (E) RIP assay. (F) CLIP assay. (G) m6A-IP assay. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. CLIP, CrossLinking immunoprecipitation; READ, rectal adenocarcinoma; RIP, RNA immunoprecipitation; SD, standard deviation; TCGA, The Cancer Genome Atlas.