Fig. 4: Nuclear export of lnc668 depended on YTHDC1 phase separation.

a Immunofluorescence demonstrated that treatment with the phase separation inhibitor 1,6-hexanediol resulted in a reduction in nuclear condensates in the TGF-β1 and normal groups. b The EGFP group did not exhibit nuclear punctate structures. By contrast, cells overexpressing EGFP-YTHDC1 displayed numerous visible nuclear punctate structures. After 1,6-hexanediol addition, the nuclear punctate structures significantly reduced. c Time-lapse live-cell imaging demonstrated that EGFP-YTHDC1 nuclear droplets exhibited rapid phase separation dynamics. Two adjacent droplets fused within 60 s. d FRAP experiments revealed rapid fluorescence recovery in a photobleached region of a nuclear punctate structure in the overexpressed EGFP-YTHDC1 group. The fluorescence intensity in the bleached area recovered to 50% of the initial intensity within 14.2 s. e FRAP confirmed that nuclear speckled structures did not form and the selected fluorescent region was photobleached in the overexpressed EGFP-YTHDC1-Δ274-294 group. The fluorescence in the bleached area did not recover even after 140 s. f Rescue experiments confirmed that lnc668 expression decreased in the si-YTHDC1 group. YTHDC1 overexpression promoted lnc668 expression and reversed the inhibitory effect of si-YTHDC1, whereas YTHDC1-Δ274-294 overexpression did not promote lnc668 expression and did not reverse the inhibitory effect of si-YTHDC1. g RNA pull-down experiments showed that the YTHDC1-Δ274-294 mutant failed to bind both lnc668 WT probe and lnc668 m⁶A probe. h Nuclear-cytoplasmic separation experiments confirmed that after si-YTHDC1 expression, cytoplasmic lnc668 expression decreased and nuclear lnc668 expression increased. Wild-type YTHDC1 overexpression increased cytoplasmic lnc668 expression and decreased nuclear lnc668 expression, reversing the inhibitory effect of si-YTHDC1. However, YTHDC1-Δ274-294 overexpression did not increase lnc668 expression in the cytoplasm or decrease lnc668 expression in the nucleus, failing to reverse the inhibitory effect of si-YTHDC1. i RNA-FISH confirmed that si-YTHDC1 reduced lnc668 expression, with a more significant decrease in the cytoplasm than in the nucleus. Exogenous YTHDC1-WT overexpression after the knockdown of endogenous YTHDC1 led to increased lnc668 expression in the cytoplasm and decreased lnc668 expression in the nucleus, reversing the inhibitory effects of si-YTHDC1. However, YTHDC1-Δ274-294 overexpression did not enhance lnc668 expression in the cytoplasm and was unable to counteract the inhibitory effect of si-YTHDC1.