Preincubation with G3Ia or G3Ib prevents the formation of stress granules and inhibits the accumulation of TDP-43 into stress granules. (A) Schematic showing the preincubation paradigm used in B–E and I. 50 μM of indicated compounds was added to cells for 20 min, followed by exposure to 250 μM NaAsO₂ stress and live cell imaging to monitor stress granule formation. (B) Representative images of G3BP1-GFP signal in U2OS cells after 20 min 250 μM NaAsO₂. Scale bar, 40 μm. (C) Quantification of cells as in B showing the percentage of stress granule area per cell. (D) Representative images of G3BP1-GFP signal in HeLa cells after 20 min of 250 μM NaAsO₂. Scale bar, 40 μm. (E) Quantification of cells in D showing the percentage of stress granule area per cell. (F) Representative images of immunofluorescent staining of additional stress granule markers (eIF3η, PABPC1) in cells pre-treated with 50 μM G3I compounds and then exposed to 250 μM NaAsO2 for 30 min. Scale bar, 20 μm. (G) Schematic showing the preincubation paradigm used in H. 50 μM of indicated compounds was added to U2OS cells for 20 min, followed by exposure to 250 μM NaAsO₂ stress, followed by fixation and immunofluorescence for G3BP1 and TDP-43. Vehicle and unstressed cells were used as controls. (H) Representative images of immunofluorescence in U2OS cells after 30 min 250 μM NaAsO₂. Scale bars, 20 and 3 μm (inset). (I) Representative images of mRuby3-G3BP1 and GFP-TDP-43 signal in U2OS cells after pretreatment with compound followed by 30 min 250 μM NaAsO₂. Arrowheads indicate puncta positive for G3BP1 and TDP-43; scale bars, 20 and 3 μm (inset). Error bars represent mean ± SEM in all graphs.