Fig. 7

YTHDF1 promoted HINT2 translation. a Reduction in m⁶A modification at specific regions of HINT2 transcripts, as assessed by gene-specific YTHDF1, YTHDF2 and YTHDF3 RIP-qPCR assays, in PIG1 cells. The value obtained for the IgG was set to 1. Error bars indicate the mean ± SEM, n = 3, **p < 0.01. b Model showing RNA probes used in RNA pull-down assays. c RNA pulldown of endogenous YTHDF1 proteins from HEK293T nuclear extract using synthetic HINT2 RNA fragments with or without m⁶A modifications. Images are representative of 3 independent experiments. d Western blotting (Top) and real-time PCR (Bottom) were performed to elucidate HINT2 expression in PIG1 cells with or without YTHDF1 knockdown. The HINT2 and YTHDF1 signals were quantified and normalized to that of GAPDH. Error bars indicate the mean ± SEM, n = 3. e, f Western blotting (Top) and real-time PCR (Bottom) were performed to elucidate HINT2 expression in PIG1 cells with or without YTHDF1 overexpression. The HINT2 and YTHDF1 signals were quantified and normalized to that of GAPDH. Error bars indicate the mean ± SEM, n = 3. g Dual luciferase reporter assays showing the effect of ALKBH5 on HINT2 mRNA reporters with either wild-type or mutated m⁶A sites. Error bars indicate the mean ± SEM, n = 3, ***p < 0.001. h Polysome profiling assays. The fractionation of lysates from PIG1 cells with or without METTL3 knockdown is shown on the top. RNAs in different ribosome fractions were extracted and subjected to qPCR analysis; data are shown on the bottom as the mean ± SD. n = 3, *p < 0.05; **p < 0.01; ***p < 0.001. i Model showing how reduced m⁶A methylation alters HINT2 translation to contribute to tumor progression