USP10 blocks SG assembly downstream of translational arrest. (A) Tet-on GFP-USP10 U2OS-WT cells were treated with increasing amounts of tetracycline for 22 h and then treated with 100 µM AS for 1 h and fixed and stained with SG markers (FMRP [FMR1], red; eIF3b, blue). Bar, 25 µm. Insets zoomed 1.3× with separated colors. The percentage of cells with SGs is indicated below each panel; n = 3. (B) Tet-on GFP-USP10 was uninduced (1–3) or induced with doxycycline (Dox) for 24 h (4–6) before indicated 1-h drug treatments. Cells were treated with 100 µM AS, (1 and 4), 20 µM CZ, (2 and 5), or 50 nM Pat A (3 and 6) and then stained for G3BP1 (red) and eIF3b (blue). Bar, 10 µm. (C) Polysome profiles obtained from uninduced tet-on GFP-USP10 cells as in B (1–3) and treated with control (green), 100 µM AS (red), or 20 µM CZ (blue); n = 3. (D) Polysome profiles of doxycycline-induced tet-on GFP-USP10 cells, treated as in B (4–6); n = 3.