Figure S3. SGs contain polyadenylated... | Rockefeller University Press

Figure S3.

$SGs contain polyadenylated mRNAs, and SG area and number are not substantially altered when VCP or the proteasome are inhibited, or when pathogenic VCP alleles are expressed during arsenite stress.(A) U-2 OS cells stably expressing the SG marker protein GFP-G3BP1 (green) were stressed for 45 min with 0.5 mM sodium arsenite (As) in the presence or absence of DMSO (0.1%), MG132 (10 µM), DBeQ (10 µM), or DBeQ (10 µM) and puromycin (Puro; 10 µg/ml), and then FISH was performed with oligo(dT)-Cy3 probes to detect polyadenylated transcripts (red). Nuclei are stained with DAPI (blue); these cells were also assessed for AHNAK mRNAs by smFISH (Fig. 2 A). Shown are representative maximum intensity projections of 25 z-stacks acquired at 100× on a DeltaVision microscope. Scale bars, 10 µm (whole cell) or 5 µm (magnified panels). Results represent n = two independent experiments. (B and C) Cells were treated as described in A, and the relative distribution of SG areas (B) and the number of SGs per cell (C) were determined using ImageJ/Fiji in the images used to quantify the fraction of AHNAK mRNAs (B) and both AHNAK and NORAD RNAs (C) colocalized with SGs (Fig. 2 and Fig. 3). For cells expressing VCP alleles (Fig. 7), results represent n = three independent experiments with average number of SGs per cell ± SEM shown in C. Student’s t test was done to assess significance, with *, P < 0.05 and ****, P < 0.001. Exact P values and source data are provided in Table S1.$

SGs contain polyadenylated mRNAs, and SG area and number are not substantially altered when VCP or the proteasome are inhibited, or when pathogenic VCP alleles are expressed during arsenite stress. (A) U-2 OS cells stably expressing the SG marker protein GFP-G3BP1 (green) were stressed for 45 min with 0.5 mM sodium arsenite (As) in the presence or absence of DMSO (0.1%), MG132 (10 µM), DBeQ (10 µM), or DBeQ (10 µM) and puromycin (Puro; 10 µg/ml), and then FISH was performed with oligo(dT)-Cy3 probes to detect polyadenylated transcripts (red). Nuclei are stained with DAPI (blue); these cells were also assessed for AHNAK mRNAs by smFISH (Fig. 2 A). Shown are representative maximum intensity projections of 25 z-stacks acquired at 100× on a DeltaVision microscope. Scale bars, 10 µm (whole cell) or 5 µm (magnified panels). Results represent n = two independent experiments. (B and C) Cells were treated as described in A, and the relative distribution of SG areas (B) and the number of SGs per cell (C) were determined using ImageJ/Fiji in the images used to quantify the fraction of AHNAK mRNAs (B) and both AHNAK and NORAD RNAs (C) colocalized with SGs (Fig. 2 and Fig. 3). For cells expressing VCP alleles (Fig. 7), results represent n = three independent experiments with average number of SGs per cell ± SEM shown in C. Student’s t test was done to assess significance, with *, P < 0.05 and ****, P < 0.001. Exact P values and source data are provided in Table S1.