Extended Data Fig. 10: O-GlcNAcylation of YTHDF1 increased the dynamic nature of stress granules.
From: O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins
a–e, FRAP analysis of mCherry-WT-YTHDF1/YTHDF1-Mut and EGFP-G3BP1 treated with 0.6 M sorbitol stress in Ythdf1/3 knockdown HeLa. YTHDF1-Mut shows lower and slower recovery in fluorescence than WT-YTHDF1 both in YTHDF1 itself and SGs (G3BP1 as marker). Scale bars: 1 μm. Error bar, mean ± s.d., n = 3 SGs per condition from 3 independent experiments. Significance was calculated using two-sided t-test. The white circles represent the photobleaching region. (f, g) Time lapse imaging of mCherry-WT-YTHDF1/YTHDF1-Mut and EGFP-G3BP1 released from 0.6 M sorbitol stress in living Ythdf1/3 knockdown HeLa cells. Scale bars: 10 μm. Error bar, mean ± s.d., n = 7, 6 cells for WT and Mut group from 3 independent experiments. Significance was calculated using two-sided t-test. (h–j) FRAP analysis of Cy3-m6A-RNA probe, BFP-G3BP1 and EGFP–YTHDF1 or O-GlcNAcylated EGFP–YTHDF1 in the droplets in vitro. Scale bars: 5 μm. Error bar, mean ± s.d., n = 3 droplets per condition from 3 independent experiments. Significance was calculated using a two-sided t-test. OG denoted O-GlcNAcylated form by OGT reaction. The white circles represent the photobleaching region.
