Treatment with G3I compounds modifies stress granules formed in response to expression of disease-causing mutant proteins. (A) U2OS cells with TdTomato-tagged endogenous G3BP1 (red) were transfected with VCP A232E (green) for 24 h, then treated with 50 μM G3I compound for 30 min. Shown are representative images of cells before (left) and after (right) addition of G3Ia or G3Ia′. Arrows indicate G3BP1-positive puncta. Scale bar, 10 μm. (B) Quantification of cells as in A showing the percentage of stress granule dissolution as assessed by TdTomato imaging. Automated puncta tracking was used for G3Ia; manual blinded cell tracking was used for G3Ib. (C) U2OS cells with TdTomato-tagged endogenous G3BP1 (red) were transfected with FUS R495X (green) for 24 h, then treated with 50 μM G3I compound for 30 min. Shown are representative images of cells before (left) and after (right) the addition of G3Ia or G3Ia′. Arrows indicate G3BP1- or FUS R495X-positive puncta. Scale bar, 10 μm. (D) Quantification of cells as in C showing the percentage of puncta dissolution for G3BP1-positive and FUS R495X-positive puncta. (E and F) U2OS cells with TdTomato-tagged endogenous G3BP1 were pretreated with 50 μM G3I compound or vehicle for 20 min prior to transfection with FUS R495X for 24 h. Using automated analysis pooled from 10 transfected wells per treatment group, the percentage of cytoplasm containing G3BP1 puncta (E) and FUS R495X puncta (F) was quantified in transfected cells. Error bars represent the mean cytoplasmic puncta area per cell ± SEM. *P < 0.1, **P < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test.