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. Author manuscript; available in PMC: 2022 Feb 5.
Published in final edited form as: Science. 2021 Aug 5;372(6549):eabf6548. doi: 10.1126/science.abf6548

Fig. 4. VCP regulates disassembly of stress granules.

Fig. 4.

(A to C) Top: U2OS/GFP-G3BP1 were treated with DMSO or bafilomycin A1 (BafA1) for 18 hours before live imaging. Scale bar, 20 μm. Bottom: GFP signals were monitored at 30-s intervals at 37°C for 2 min, 43°C for 30 min (A), 60 min (B), or 90 min (C), and 37°C for 88 min (A), 118 min (B), or 148 min (C) to count cells with two or more stress granules from three technical replicates [DMSO n = 56, BafA1 n = 58 in (A); DMSO n = 56, BafA1 n = 58 in (B); DMSO n = 52, BafA1 n = 59 in (C)]. Error bars indicate SEM. ****P < 0.0001 (Mantel-Cox test). (D and E) U2OS/GFP-G3BP1 cells were treated with DMSO or CB-5083 (1 hour) (D) or transfected with nontargeting (NT) or VCP siRNA (E) before live imaging. GFP signals were monitored at 30-s intervals at 37°C for 2 min, 43°C for 60 min, and 37°C for 118 min to count the cells with two or more stress granules [DMSO n = 40, CB-5083 n = 47 in (D); NT siRNA n = 44, VCP siRNA n = 53 in (E)]. Scale bars, 50 μm (D), 20 μm (E). Error bars indicate SEM. ****P < 0.0001 (Mantel-Cox test). (F) Immunoblot of U2OS cells transfected with NT or VCP siRNA from three biological replicates. Error bars indicate SEM. **P < 0.01 (Student’s t test). (G) U2OS/GFP-G3BP1 cells were transfected with pmCherry-N1, VCP WT-mCherry, VCP R155H-mCherry, or VCP A232EmCherry. Scale bar, 10 μm. (H) GFP signals of mCherry-positive cells were monitored at 60-s intervals at 37°C for 2 min, 43°C for 60 min, and 37°C for 88 min to count cells with two or more stress granules from three biological replicates (control n = 23, VCP WT n = 29, VCP R155H n = 26, VCP A232E n = 39). Error bars indicate SEM. **P < 0.01 (Mantel-Cox test). (I) U2OS cells expressing photoactivatable G3BP1 (G3BP1-PAGFP) were transfected with NT or VCP siRNA. (J) Intensities of GFP signals within activated ROIs were monitored at 200-ms intervals from three technical replicates. ****P < 0.0001 (ANOVA with Sidak’s test). (K) Immunoblots of U2OS cells expressing G3BP1-PAGFP transfected with NT or VCP siRNA from three biological replicates. ***P< 0.001 (Student’s t-test).