Pathogenic VCP alleles increase recruitment of AHNAK mRNAs, but not NORAD lncRNAs, to SGs. U-2 OS cells stably expressing the SG marker mRuby2-G3BP1 were either untransfected (None) or transiently transfected with empty vector peGFP-N1 (eGFP), WT VCP fused to eGFP (VCP(WT)-eGFP), or VCP alleles (VCP(A232E)-eGFP and VCP(R155H)-eGFP). Cells were stressed for 45 min with 0.5 mM arsenite (As) and then fixed, and smFISH was performed to detect AHNAK mRNA (top) or NORAD lncRNA (bottom). Representative photomicrographs for are shown at left, with RNAs shown in white, SGs in red, and eGFP in green. Images were acquired on a DeltaVision microscope at 100×, and maximum intensity projections of 25 z-stacks are shown. Scale bars, 10 µm (whole cell) or 5 µm (magnified panels). At right, average percent AHNAK mRNA or NORAD lncRNA ± SEM that colocalize with SGs. Three independent experiments were done with individual dots representing one frame, and only cells expressing eGFP were counted for all transfected conditions. For AHNAK, None n = 11 frames, GFP n = 12 frames, VCP(WT) n = 11 frames, VCP(A232E) n = 12 frames, VCP(R155H) n = 13 frames; for NORAD, None n = 8 frames, GFP n = 10 frames, VCP(WT) n = 10 frames, VCP(A232E) n = 9 frames, VCP(R155H) n = 9 frames. Student’s t test was done to assess significance between GFP and VCP(WT), VCP(A232E), or VCP(R155H) for AHNAK and NORAD, with *, P ≤ 0.05. Exact P values and source data are provided in Table S1.