Fig. 5.

Nuclear export of SOX2 is a part of 10580-dependent GIC death. (A) O-GlcNAcylation of SOX2 through the OGT/UDP-GlcNAc reaction. (B) Proportion of SOX2+ cells among E6 and E16 GICs cultured in the presence of 10580 alone or in combination with UDP-GlcNAc. (C) Dose-dependent effects of UDP (open circle) and UDP-GlcNAc (closed circle) in 10580 (10 µM)-treated E6 and E16 GICs. (D) O-GlcNAcylation analysis of SOX2 in E6 GIC that were cultured in DMSO alone (N), 10580 alone (–), 10580+UDP-GlcNAc (GlcNAc), or 10580+UDP. Immunoprecipitated cell lysates using anti-FLAG M2 antibody were analyzed by western blotting (upper panel). O-GlcNAcylated SOX2 level shown in upper panels was presented as fold change against FLAG (lower panel). Protein quantification was performed using ImageJ software. (E) Proportion of nuclear SOX2-retaining GICs expressing control, SOX2 wt, SOX2 K75A, or SOX2 S248A. (F) Proportion of survival/proliferating 10580-treated GICs in the presence (black column) or absence (white column) of the pan-caspase inhibitor Z-VAD-FMK. (G) Proportion of nuclear SOX2+ GICs that were cultured in DMSO, 10580, or 10580+Z-VAD for 2 days. ns: not significant. All experiments were repeated at least 3 times with similar results. Error bar: ±SD. Statistical significance was determined by t-test. *P < 0.05, **P < 0.01, ***P < 0.001.