Fig 1: hSSB1 is organized into cytoplasmic foci upon oxidative stress, colocalizing with stress granules(A) Representative confocal microscopy images (n = 3 independent experiments) of immunostained, untreated or H2O2-treated (300 μM, 2 h) HeLa cells. Blue channel shows nuclear DAPI stain, green and red channels show endogenous hSSB1 and G3BP1 stress granule marker, respectively. Merge image was created from hSSB1 and G3BP1 channels; yellow cytoplasmic foci indicate enrichment of hSSB1 in G3BP1-positive SGs.(B) Relative nuclear hSSB1 intensity decreases upon H2O2 treatment (Mann-Whitney-test; ∗ indicates significance; ‘ns’, not significant; p < 0.05; RFU, relative fluorescence units). Dots indicate individual nuclear intensities.(C) Top row, magnified confocal images of a H2O2-treated HeLa cell (cf. panel A), with hSSB1 and G3BP1 showing colocalized enrichment in cytoplasmic stress granules. Bottom row, 3D reconstructed images of the same ROI from confocal z stack images (16 slices, 3.6 μm optical sectioning). Green and red channels show endogenous hSSB1 and G3BP1 stress granule marker, respectively; yellow color indicates colocalization.(D) Manders’ split coefficients (left) and object based colocalization (right) of detected cytoplasmic hSSB1 and G3BP1 condensates. Dots indicate individual data derived from an image.
Fig 2: The effect of proline in G3BP–G3BP interaction (A) and ssDNA–ssDNA interaction (B) in solution and the proposed mechanism for the modulation of AAs on the formation of SGs (C). The SGs are formed in the cytosol as a result of complex interactions among an assembly of proteins (including scaffold proteins which are crucial for the formation of SGs and a large variety of IDP) as well as many RNA in response to stress. The presence of AAs is proposed to render net attractive forces between these SG components weaker and thus modulate the formation of SGs. The modulation effect is composed of three aspects: i) lower SG formation rate; ii) smaller SG saturating number; and iii) less SG coalescence.
Fig 3: Live cell microscopy of GFP-tagged G3BP1 showing the effect of proline and glutamine (200 mM) on the formation process of SGs in U2OS cells. (A) The pretreatment for 2 h, the cotreatment, and the posttreatment (15 mins after the addition of NaAs) with 200 mM proline all suppressed the formation of SGs compared to positive control (no exogenous AAs were added) by live cell microscopy of U2OS cells with GFP tagged G3BP1 (the coalescence events indicated in the red squares). (B) Time series of the number of SGs per cell after the addition of NaAs including positive control (no proline treatment) and 200 mM proline pre-/co-/posttreatment (indicated as lag, growth and coalescence phases). (C) Time series of the number of SGs per cell after the addition of NaAs including positive control (no glutamine treatment) and 200 mM glutamine pre-/co-/posttreatment (related live cell images in SI Appendix, Fig. S9). (Scale bar, 10 μm.)
Fig 4: hSSB1 is enriched in stress granules in response to various cellular stressors(A) Representative confocal microscopic images (n = 3 independent experiments) of immunostained HeLa cells treated with various stress agents (H2O2: 1 mM, 2 h; KBrO3: 30 mM, 2 h; NaAsO2: 0.5 mM, 2 h; Menadione sodium bisulfite: 100 μM, 4 h; DTT: 1 mM, 2 h; Etoposide: 100 μM, 2 h). Blue channel shows nuclear Hoechst stain; green and red channels show endogenous hSSB1 and G3BP1 stress granule marker, respectively. Merged image was created from all three channels; yellow cytoplasmic foci indicate enrichment of hSSB1 in G3BP1-positive SGs. Scale bar: 20 μm.(B) Besides H2O2, NaAsO2, menadione sodium bisulfite (mena), and DTT triggered a decrease in nuclear hSSB1 intensity, while KBrO3 and etoposide (eto) did not. Changes in nuclear hSSB1 intensities (RFU, relative fluorescence units) were normalized to appropriate serum deprivation controls. Dots indicate individual nuclear intensities (Kruskal-Wallis test with Dunn’s post-hoc test, ∗ indicates significant difference, ‘ns’ not significant, p < 0.05).(C) Besides H2O2, NaAsO2, menadione sodium bisulfite, and DTT induced robust SG formation, while KBrO3 and etoposide did not. Changes in the average number of SGs per cell are shown. Dots represent average SG numbers derived from individual images (Kruskal-Wallis test with Dunn’s post-hoc test, ∗ indicates significant difference, p < 0.05).(D) Manders’ split coefficients show colocalization of hSSB1 and G3BP1 condensates upon treatment with H2O2, NaAsO2, menadione sodium bisulfite, and DTT. Dots represent Manders’ coefficients derived from individual images.
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