Fig 1: Raloxifene inhibits dissolution of hypoxia-induced SGs in glioblastoma cells and does so in a dose-dependent manner.A Raloxifene dose response curve. U251 glioblastoma cells were treated with raloxifene at doses ranging from 0 to 60 μM for 1 h prior to a 2 h incubation ± hypoxia (<1% O2). Cells were allowed to recover for 1 h in normoxia and then fixed and stained for SG markers TIAR and FMRP. Percentage of cells with SGs were counted manually; 10 fields of view ~50 cells per field. Data is presented as the mean of triplicates ± SEM, unpaired t-test ***p < 0.001. B Immortalized GBM U251 cells and C primary GBM U3024 cells were treated with 40 μM raloxifene or vehicle control (DMSO) for 1 h followed by a 2 h (U251) or 1 h (U3024) incubation ± hypoxia (<1% O2). Cells were then fixed immediately (0 min) or allowed to recover (30 min) in normoxia before being fixed and stained for SG markers TIAR (green) and G3BP2 (red). DNA was counterstained with DAPI (blue). Scale bars = 10 microns.
Fig 2: Hypoxic stress responses are activated in low-grade astrocytoma and GBM and correlate to poor outcomes.A TCGA database was interrogated with GlioVis for mRNA expression levels of genes involved in activation of the ISR (PERK, GCN2), in nucleating SGs (G3BP1) and terminating the ISR and triggering SG disassembly (GADD34) in grade II, III, and IV astrocytoma. Statistical analysis using Tukey’s honest significant difference and performed on GlioVis, ***p < 0.001, **p < 0.01, and *p < 0.05. B Kaplan–Meir survival curves for high and low mRNA expression of PERK, GCN2, G3BP1, GADD34 in low-grade astrocytoma. p-values adjusted for multiple comparisons. C Graphical representation of IHC cytoplasmic granular scoring of a TMA consisting of 90 GBM cores from 45 patients stained with SG markers G3BP2 and TIAR. Grading was assigned 0–4, no staining, minority of cells, ~50% of cells, majority of cells or all cells (excluding vascular endothelium), respectively. Example of staining grades on left panel. D Examples of IHC G3BP2 and TIAR-stained GBM and normal cortex showing cytoplasmic granular staining (blue arrowheads). The vascular endothelium encasing tumor is visible (red arrow) with negative staining of G3BP2 and TIAR. White boxes denote magnified areas. Black line represents 100 μm. E IHC example of GBM core stained for TIAR and G3BP2 demonstrating necrosis (right of orange dotted line) and increased granular staining in the cells adjacent to the necrotic core. Black boxes magnified and granular punctate demonstrated by blue arrow heads. Black line is 100 μm.
Fig 3: UV-induced SG formation is dependent on G3BP1. (A–D) G3BP1, G3BP2, and control PKR knockout cells were generated using CRISPR/Cas9. (A) PKR, G3BP1 and G3BP2 expression in CRISPR knockout cells and eIF2α phosphorylation in response to 20 mJ/cm2 UV light (UV) were analysed by western blotting. Staining for actin was used as loading control. (B) UV-induced SG formation (20 mJ/cm2) was analysed by immunofluorescence microscopy in parental U2OS cells (U2OS) and CRISPR knockout cell lines lacking G3BP1 (ΔG3BP1), G3BP2 (ΔG3BP2) or PKR (ΔPKR), and stained for G3BP1 (red) and TIAR (green). Nuclei were stained with Hoechst dye (blue). (C) UV-induced SG formation was quantified (mean±s.d.) from immunofluorescence microscopy staining represented in B (n=3). *P<0.05 (two-way ANOVA followed by Tukey's multiple comparisons test). (D) Selenite-induced SG formation was analysed by immunofluorescence microscopy in parental U2OS cells (U2OS) and CRISPR knockout cell lines lacking G3BP1 (ΔG3BP1) or G3BP2 (ΔG3BP2) stained for G3BP1 (red) and G3BP2 (green). Nuclei were stained with Hoechst dye (blue). (E,F) Expression of the EGFP–USP10(1-40) fusion protein or EGFP control was induced in U2OS cells stably transduced with lentiviral constructs encoding these proteins. After 48 h, cells were left untreated (−), or were treated with UV light (UV, 20 mJ/cm2) or sodium arsenite (As). (E) Phosphorylation status of GCN2 and eIF2α post-treatment was analysed by western blotting, as well as the expression of EGFP–USP10(1-40) fusion protein or EGFP control. Staining for actin was used as loading control. (F) SG formation was analysed by immunofluorescence microscopy staining for G3BP1 (red) and TIAR (blue) in these GFP-positive cells (green). Numbers indicate the percentage of cells with SGs quantified from at least three random fields of view containing >100 cells (mean values, n=2).
Fig 4: Raloxifene delays SG dissolution for up to 2 h post-hypoxia.Quantitative analysis of SGs. Immortalized U251 A–C and primary U3024 GBM cells D and E were treated with 40 μM raloxifene or vehicle control (DMSO) for 1 h before being subjected to either 2 h (U251) or 1 h (U3024) of hypoxia (<1% O2). Cells were fixed at various times post-hypoxia and stained for SG markers TIAR and G3BP2. Cells were also stained with wheat germ agglutinin to denote cellular membranes and DAPI to identify nuclei. Percentage of cells with SGs and average number of SGs per cell (in those cells containing SGs) or SG intensity were then quantified from correlative TIAR and G3BP2 staining using an automated image analysis pipeline in CellProfiler. Data is presented as the mean of triplicates ± SEM, unpaired t-test. *p < 0.05; **p < 0.01***; and p < 0.001.
Fig 5: G3BP1 knockout partially reverses raloxifene-induced delay of SG dissolution.A U251 cell lines stably expressing CRISPR gRNAs targeting G3BP1 or G3BP2 along with non-targeting (nt) gRNAs were established using a lentiviral-based system. B Untransduced (WT) U251 and nt gRNA control (NTC) cells along with G3BP1 and G3BP2 knockout clones were treated with 40 μM raloxifene or DMSO vehicle control prior to 2 h of hypoxia (<1% O2). Cells were fixed either immediately (0 min) or 30 min post-hypoxia. Cells were stained for CellProfiler analysis as previously described, and average number of SGs per cell (in those cells containing SGs) were quantified from correlative TIAR and FMRP staining in CellProfiler. Data is presented as the mean of triplicates ± SEM. C Representative immunofluorescence images. Cells were stained for SG markers TIAR (green) and FMRP (red), and DNA was counterstained with DAPI (blue) immediately (0 min) or 30 min post-hypoxia. Scale bars = 10 microns.
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