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. 2022 Dec 27;42(3):e111802. doi: 10.15252/embj.2022111802

Figure 2. Compromised nuclear UPS in stress granule‐deficient cells.

Figure 2

  • A, B
    Representative images of stress granule proficient (siControl) and deficient (siG3BP1/2) MelJuSo cells expressing the nuclear UPS reporter NLS‐GFP‐CL1 (A) and the cytoplasmic UPS reporter NES‐GFP‐CL1 (B). The cells were left untreated (− HS), exposed to 43°C for 30 min (HS), or exposed to 43°C for 30 min and followed by 4 h recovery (HS + 4 h Rec.). Nucleolar accumulation of NLS‐GFP‐CL1 during the recovery is indicated by arrows in (A). Scale bar, 20 μm.
  • C, D
    Quantifications of the mean cellular GFP fluorescence intensities of (A) and (B) normalized to untreated control cells (siControl ‐ HS). The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n = 3 independent experiments, > 1,000 cells analyzed per condition, Kruskal‐Wallis test, **P < 0.01, n.s.—not significant).
  • E
    Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS‐GFP‐CL1 in control (siControl, left panel) and G3BP1/2‐depleted (siGRBP1/2, right panel) MelJuSo cells exposed to a 30 min 43°C heat shock, followed by 4 h at 37°C (Recovery). Nucleolar accumulation of NLS‐GFP‐CL1 is indicated by arrows. Scale bar, 10 μm.
  • F
    Quantification of the nucleolus/nucleoplasm ratios of NLS‐GFP‐CL1 intensities in images of the experiment shown in (E). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n = 3 independent experiments, 100 cells analyzed per condition, Kruskal‐Wallis test, **P < 0.01).