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. 2024 May 20;43(13):2759–2788. doi: 10.1038/s44318-024-00120-6

Figure EV2. Ubiquitin regulates oligomycin-induced P-bodies dynamics.

Figure EV2

(A) HEK293T cells were treated with 2-DG or/and oligomycin for 2 h. The ubiquitination level of the proteins was detected by immunoblotting. (B) Immunoblot of HEK293T cells treated with TAK-243 or PYR-41 (2 h). (C, D) HEK293T cells were treated with TAK-243 or PYR-41 (1 h) in the presence or absence of oligomycin (1 h) and immunolabeled for DCP1A. Representative images are shown in (C). Scale bar, 5 μm. The number of P-bodies within each cell is plotted in (D) (n = 50). Error bars indicate SEM. ****P < 0.0001 (two-way ANOVA). (E) Immunoblot of HCT116 cells treated with TAK-243 or PYR-41 (2 h). (F, G) HCT116 cells were treated with TAK-243 or PYR-41 in the presence or absence of oligomycin (1 h) and immunolabeled for LSM14A. Representative images are shown in (F). Scale bar, 5 μm. The number of P-bodies within each cell is plotted in (G) (n = 50). Error bars indicate SEM. ****P < 0.0001 (two-way ANOVA). (H, I) HEK293T cells were treated with MG132 or RA190 (1 h) in the presence or absence of oligomycin (1 h) and immunolabeled for DCP1A. Representative images are shown in (H). Scale bar, 5 μm. The number of P-bodies within each cell is plotted in (I). Error bars indicate SEM. ns: no significance (two-way ANOVA). (J) Immunoblot of control and UBB-knockdown HEK293T cells. (K, L) Control and UBB-knockdown HEK293T cells were treated with oligomycin (1 h) or puromycin (1 h), and stained for DCP1A and DAPI. Representative images are shown in (K). The number of P-bodies within each cell is plotted in (L) (n = 50). Error bars indicate SEM. **P < 0.01, ****P < 0.0001 (two-way ANOVA). (M, N) HEK293T cells transfected with vector, Ub, UbK48R or UbK63R were stained for DCP1A and DAPI. Representative images are shown in (M). The number of P-bodies within each cell is plotted in (N) (n = 50). Scale bar, 5 μm. Error bars indicate SEM. Ns: no significance, ****P < 0.0001 (Student’s t test). (O, P) FRAP analysis of GFP-LSM14A in HEK293T cells transfected with vector or Ub. White arrows indicate the target droplets for FRAP. Scale bar, 5 μm. n = 3, Error bars indicate SEM. **P < 0.01 (two-way ANOVA).