Figure 2.
G3Ia and G3Ib disrupt in vitro condensation of RNA, G3BP1, and caprin 1. (A–D) Lysates from U2OS cells stably expressing G3BP1-GFP were collected, incubated with increasing concentrations of G3I compound, immunoprecipitated for GFP, and separated by SDS-PAGE. Blots were probed for GFP and endogenous caprin 1 (A and B) or endogenous USP10 (C and D). GAPDH was used as a loading control. Densitometry from n = 3 blots was used to generate graphs (B and D); error bars represent mean ± SD. *P = 0.0495, **P = 0.0011, ***P = 0.0002 by one-way ANOVA with Dunnett’s multiple comparisons test. (E) Lysates from HEK293T cells expressing GFP-NTF2L were collected, incubated with increasing concentrations of G3I compounds or nsP3, immunoprecipitated for GFP, and separated by SDS-PAGE. Blots were probed for GFP, endogenous caprin 1, and endogenous USP10. Actin was used as a loading control. A representative blot is shown from n = 3 experiments. (F) Quantification of densitometry from n = 3 blots as shown in E. Error bars represent mean ± SD. ***P = 0.0006 and ****P < 0.0001 for caprin 1, **P = 0.0087 (25 μM G3Ib), **P = 0.0050 (50 μM G3Ib), **P = 0.0015 (25 μM nsP3), ***P = 0.0001 (50 μM nsP3) for USP10 by one-way ANOVA with Dunnett’s multiple comparisons test. (G) 1.5 μM G3BP1, 1.5 μM caprin 1, and 20 ng/μl total RNA were coincubated in a three-component system and co-condensation was assessed in the presence of increasing concentrations of G3I compounds. The percent inhibition of G3BP1-GFP in vitro phase separation is shown. Tables show the highest (top) and lowest (bottom) values of an individual curve, LogIC50, the slope at the steepest part of the curve (HillSlope), and IC50. Error bars represent mean ± SD, N = 3 replicates per condition. (H) 20 μM purified G3BP1 and 100 ng genomic RNA were coincubated in a two-component system and condensation was assessed in the presence of indicated doses of G3Ib or vehicle control. Condensate formation by G3BP1 and RNA was unaffected by the addition of G3Ib. Scale bar, 30 μm. (I) Cytotoxicity assay in U2OS cells treated with indicated concentrations of compounds for 24 h; inhibition of growth was measured by monitoring ATP levels read out through a luciferase signal. N = 2, both replicates are plotted. Source data are available for this figure: SourceData F2.

G3Ia and G3Ib disrupt in vitro condensation of RNA, G3BP1, and caprin 1. (A–D) Lysates from U2OS cells stably expressing G3BP1-GFP were collected, incubated with increasing concentrations of G3I compound, immunoprecipitated for GFP, and separated by SDS-PAGE. Blots were probed for GFP and endogenous caprin 1 (A and B) or endogenous USP10 (C and D). GAPDH was used as a loading control. Densitometry from n = 3 blots was used to generate graphs (B and D); error bars represent mean ± SD. *P = 0.0495, **P = 0.0011, ***P = 0.0002 by one-way ANOVA with Dunnett’s multiple comparisons test. (E) Lysates from HEK293T cells expressing GFP-NTF2L were collected, incubated with increasing concentrations of G3I compounds or nsP3, immunoprecipitated for GFP, and separated by SDS-PAGE. Blots were probed for GFP, endogenous caprin 1, and endogenous USP10. Actin was used as a loading control. A representative blot is shown from n = 3 experiments. (F) Quantification of densitometry from n = 3 blots as shown in E. Error bars represent mean ± SD. ***P = 0.0006 and ****P < 0.0001 for caprin 1, **P = 0.0087 (25 μM G3Ib), **P = 0.0050 (50 μM G3Ib), **P = 0.0015 (25 μM nsP3), ***P = 0.0001 (50 μM nsP3) for USP10 by one-way ANOVA with Dunnett’s multiple comparisons test. (G) 1.5 μM G3BP1, 1.5 μM caprin 1, and 20 ng/μl total RNA were coincubated in a three-component system and co-condensation was assessed in the presence of increasing concentrations of G3I compounds. The percent inhibition of G3BP1-GFP in vitro phase separation is shown. Tables show the highest (top) and lowest (bottom) values of an individual curve, LogIC50, the slope at the steepest part of the curve (HillSlope), and IC50. Error bars represent mean ± SD, N = 3 replicates per condition. (H) 20 μM purified G3BP1 and 100 ng genomic RNA were coincubated in a two-component system and condensation was assessed in the presence of indicated doses of G3Ib or vehicle control. Condensate formation by G3BP1 and RNA was unaffected by the addition of G3Ib. Scale bar, 30 μm. (I) Cytotoxicity assay in U2OS cells treated with indicated concentrations of compounds for 24 h; inhibition of growth was measured by monitoring ATP levels read out through a luciferase signal. N = 2, both replicates are plotted. Source data are available for this figure: SourceData F2.

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