Treatment of cells depleted of LTN1 or NEMF with VCP or proteasome inhibitors does not further deplete AHNAK mRNAs from SGs during arsenite stress. U-2 OS cells expressing the SG marker G3BP1-GFP were depleted of LTN1 (siLTN1) or NEMF (siNEMF) using siRNAs or transfected with nonspecific control siRNAs (siNS). Cells were stressed with 0.5 mM arsenite (As) for 45 min with 0.1% DMSO (As + DMSO), 10 µM MG132 (As + MG132), 10 µM MG132 with 10 µg/ml puromycin (As + MG132 + Puro), 10 µM DBeQ (As + DBeQ), or 10 µM DBeQ with 10 µg/ml Puro (As + DBeQ + Puro), fixed, and smFISH for AHNAK mRNA was performed. Samples were imaged at 100× on a DeltaVision microscope. Representative image maximum intensity projections of 25 z-stacks are presented (top), with scale bars representing 10 µm (whole cells) or 2 µm (magnified panels), with G3BP1-GFP in green, AHNAK mRNAs in white, and nuclei in blue. The percent AHNAK mRNAs that colocalized with SGs was determined in individual cells, and the average ± SEM is shown (bottom) from two independent experiments. For siNS, As + DMSO n = 12 cells, As + MG132 n = 13 cells, As + MG132 + Puro n = 12 cells, As + DBeQ n = 12 cells, As + DBeQ + Puro n = 12 cells; for siLTN1, n = 12 cells for all conditions; for siNEMF, n = 12 cells for all conditions. Student’s t tests were done to assess significance, with *, P ≤ 0.05; **, P ≤ 0.01; and ****, P ≤ 0.001. Exact P values and source data are provided in Table S1.