Figure 6.

A functional m⁶A-binding domain is necessary for the tumorigenic functions of YTHDC1. A) Western Blot analysis of YTHDC1 and SMAD3 in MDA-MB-231 control or YTHDC1 KO cells overexpressing either empty vector, wild type (WT), or m⁶A-binding defective mutants (W377A and W428A) of YTHDC1. B) Transwell migration and invasion assays of MDA-MB-231 control (sgNS) or YTHDC1 KO cells overexpressing either empty vector, wild type (WT), or mutant (W377A or W428A) YTHDC1 (5×10⁴ cells/well incubated for 24 hours for migration and invasion). One-way ANOVA compared to Vector/sgNS group. C) CLIP-RT-qPCR of SMAD3 bound to wild type (WT) or mutant (W377A or W428A) YTHDC1 in MDA-MB-231 cells. One-way ANOVA compared to WT group. D) Western Blot for immunoprecipitation of wild type or mutant YTHDC1 using anti-flag or control IgG antibodies in CLIP-RT-qPCR assay. (E) Dual luciferase assay in HEK293T cells transfected with WT or mutant SMAD3 3'UTR fused to a firefly luciferase (Fluc) gene. Renilla luciferase (Rluc) used as a loading control. One-way ANOVA compared to shNS+WT SMAD3 3'UTR. (F) Changes in the nuclear to cytoplasmic ratio of firefly luciferase mRNA in HEK293T cells quantified by RT-qPCR. One-way ANOVA compared to shNS+WT SMAD3 3'UTR. The data are presented as the mean ± SD; n = 3/group. NS = not significant, *P < 0.05. (G) Western Blot assay showing nuclear and cytoplasmic distribution of YTHDC1 protein in HEK293T cells and YTHDC1 KD efficiency. HDAC1 and GAPDH were used as nuclear and cytoplasmic marker, respectively.