Figure 2.
VCP and the proteasome are required for efficient release of endogenous mRNAs from ribosomes for partitioning into SGs.(A) U-2 OS cells expressing the SG marker GFP-G3BP1 were stressed with 0.5 mM sodium arsenite (As; AHNAKn = 9 cells, NORADn = 8 cells) for 45 min with or without 0.1% DMSO (As + DMSO, AHNAKn = 12 cells, NORADn = 8 cells), 10 µM MG132 (As + MG132, AHNAKn = 8 cells, NORADn = 8 cells), 10 µM DBeQ (As + DBeQ, AHNAKn = 8 cells, NORADn = 8 cells), or 10 µM DBeQ + 10 µg/ml puromycin (As + DBeQ + Puro, AHNAKn = 8 cells, NORADn = 8 cells). (B) Cells were stressed with As and cotreated as in A except in the presence or absence of CB-5083 or bortezomib (Bort). For As + DMSO AHNAKn = 21 cells, and for NORADn = 23 cells; for As + CB-5083 AHNAKn = 31 cells, and for NORADn = 22 cells; for As + CB-5083 + puromycin (Puro) AHNAKn = 25 cells and for NORADn = 17 cells; for As + Bort AHNAKn = 17 cells, and for NORADn = 21 cells. (C) Cells were stressed with As and cotreated as in A with DMSO, MG132, or Bort in the presence or absence of puromycin (n = 12 cells for all conditions). Cells were fixed, smFISH was performed to detect the mRNA AHNAK or lncRNA NORAD, and maximum intensity projections assembled from z-stacks were imaged at 100× on a DeltaVision microscope. SGs are shown in green, RNAs in white, and nuclei in blue (DAPI). Representative photomicrographs with the average percent AHNAK or NORAD RNAs colocalizing with SGs ± SEM are shown (right). Two independent experiments were performed with individual points representing a single cell, and Student’s t test was done to assess significance, with *, P ≤ 0.05; **, P ≤ 0.01; and ****, P ≤ 0.001. Scale bars, 10 µm (whole cell), 5 µm (magnified panels in A and C), or 2 µm (magnified panels in B). Exact P values and source data are provided in Table S1.

VCP and the proteasome are required for efficient release of endogenous mRNAs from ribosomes for partitioning into SGs. (A) U-2 OS cells expressing the SG marker GFP-G3BP1 were stressed with 0.5 mM sodium arsenite (As; AHNAKn = 9 cells, NORADn = 8 cells) for 45 min with or without 0.1% DMSO (As + DMSO, AHNAKn = 12 cells, NORADn = 8 cells), 10 µM MG132 (As + MG132, AHNAKn = 8 cells, NORADn = 8 cells), 10 µM DBeQ (As + DBeQ, AHNAKn = 8 cells, NORADn = 8 cells), or 10 µM DBeQ + 10 µg/ml puromycin (As + DBeQ + Puro, AHNAKn = 8 cells, NORADn = 8 cells). (B) Cells were stressed with As and cotreated as in A except in the presence or absence of CB-5083 or bortezomib (Bort). For As + DMSO AHNAKn = 21 cells, and for NORADn = 23 cells; for As + CB-5083 AHNAKn = 31 cells, and for NORADn = 22 cells; for As + CB-5083 + puromycin (Puro) AHNAKn = 25 cells and for NORADn = 17 cells; for As + Bort AHNAKn = 17 cells, and for NORADn = 21 cells. (C) Cells were stressed with As and cotreated as in A with DMSO, MG132, or Bort in the presence or absence of puromycin (n = 12 cells for all conditions). Cells were fixed, smFISH was performed to detect the mRNA AHNAK or lncRNA NORAD, and maximum intensity projections assembled from z-stacks were imaged at 100× on a DeltaVision microscope. SGs are shown in green, RNAs in white, and nuclei in blue (DAPI). Representative photomicrographs with the average percent AHNAK or NORAD RNAs colocalizing with SGs ± SEM are shown (right). Two independent experiments were performed with individual points representing a single cell, and Student’s t test was done to assess significance, with *, P ≤ 0.05; **, P ≤ 0.01; and ****, P ≤ 0.001. Scale bars, 10 µm (whole cell), 5 µm (magnified panels in A and C), or 2 µm (magnified panels in B). Exact P values and source data are provided in Table S1.

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