Stress-induced HSPA1A and HSPA1B mRNAs are not dependent on VCP to localize to SGs. (A and B) U-2 OS cells stably expressing the SG marker G3BP1-GFP were stressed for 45 min with arsenite (As; 0.5 mM; A) or heat stress (HS; 42°C; B) in the presence or absence of DMSO (0.1%), DBeQ (10 µM), or DBeQ (10 µM) plus puromycin (Puro; 10 µg/ml). Cells were fixed, and smFISH was performed to detect HSPA1A (top panels) and HSPA1B (bottom panels), and samples were imaged at 100× on a DeltaVision microscope. Representative maximum intensity projections of 25 z-stacks are shown at left with G3BP1-GFP in green, mRNAs in white, and nuclei in blue. Scale bars, 10 µm (whole cell) and 2 µm (magnified panels). The average ± SEM from two independent experiments is shown at right with individual data points shown for single cells (n = 12 cells for every condition). Student’s t test was performed to assess significance, with **, P < 0.01; ***, P < 0.005; and ****, P < 0.001. Exact P values and the source data are provided in Table S1.