Fig 1: GSK blocks SG formation induced by VRB, whereas GCN2 inhibitor blocks cortisone-induced SGs in MEFs. (A) U2OS cells treated with the PERK inhibitor GSK (40 µM) for 2 h before addition of VRB (75 µM) or VRB (75 µM) and cortisone (Cor, 300 µM) for 1 h, as indicated. SGs are stained with antibodies to detect G3BP1 (green) and eIF4B (magenta). (B) MEFs treated with a GCN2 inhibitor (GCN2IN, 4 µM) for 2 h before Cor (300 µM) addition, as indicated. SGs are labeled with antibodies to detect G3BP1 (green) and G3BP2 (magenta). (C) U2OS cells treated with GCN2 inhibitor (4 µM) and GSK (40 µM) for 2 h before addition of VRB (75 µM) and Cor (300 µM) for 1 h, as indicated. SGs are labeled for G3BP1 (green) and eIF4B (magenta). In A–C, arrowheads indicate SGs. Scale bars: 10 µm. Images are representative of three experiments. (D) Western blot analysis of eIF2α and phosphorylated eIF2α (P-eIF2α) protein levels in U2OS cells after treatments with GCN2 inhibitor (4 µM) and GSK (40 µM) for 2 h before VRB (75 µM) and Cor (300 µM) addition for 1 h, as indicated. Tubulin was used as a loading control. This experiment is representative of three separate repeats. Mean±s.d. fold change is designated under the lanes; the analysis was performed by normalizing the ratio of P-eIF2α to eIF2α for each treatment group to that of the untreated group. (E) Scheme depicting the ISR pathway and SG formation by VRB and cortisone.
Fig 2: Cortisone enhances the formation of VRB-induced SGs. (A) The formation of SGs under VRB (50–125 µM) and cortisone (Cor; 300 µM) treatment for 1 h in U2OS cells was detected using anti-eIF4B (magenta) and anti-G3BP1 (green) SG markers. Hoechst 33342 DNA stain is shown in blue. Dashed boxes indicate regions shown in the enlarged merge images on the right. Scale bar: 10 µm. (B) Quantification of the population of SG-positive U2OS cells under the different treatment conditions as in A. Data are presented as mean±s.e.m., with each circle on the bar graph indicating a biological replicate (n=3). *P<0.05; ***P<0.001 [one-way ANOVA with Tukey's post hoc test for comparing treatments with non-constant values and two-tailed one-sample t-tests (against mean values of 0 or 100) for comparing treatments with constant values (either 0% or 100%) with treatments with non-constant values]. (C) VRB and cortisone increase phosphorylated eIF2α (P-eIF2α) levels. Western blot analysis of eIF2α and P-eIF2α levels in protein extracts from U2OS cells after addition of VRB (50 µM), Cor (300 µM) or methanol (Met, 3%), and their indicated combinations, for 1 h treatments. Tubulin was used as loading control. This experiment is representative of five separate repeats. Mean±s.d. fold change in P-eIF2α is designated under the lanes; the analysis was performed by normalizing the ratio of P-eIF2α to eIF2α for each treatment group to that of the untreated group. (D,E) Frames from time-lapse movies showing SG formation in U2OS cells stably expressing GFP–IGF2B under treatment with 50 µM VRB (D; see Movie 1) or with 50 µM VRB and 300 µM Cor (E; see Movie 2). Images were acquired every 3.5 min for ∼1 h (time shown as min:s). The boxed frames (∼30 min time points) show that no SGs were formed in the VRB-treated cells, whereas SGs were observed when cortisone was added alongside VRB (arrowheads, SGs). Scale bar: 10 μm. (F) Quantification of the population of SG-positive U2OS cells under the different treatment conditions as in D and E. Data were analyzed using a two-tailed one-sample t-test against a constant mean value of 0 to compare the fraction of SG-positive cells for the two treatments at the last time point (*P<0.05). Lines represent the mean of n=30 videos, with SG-positive cells quantified at 20 time points.
Fig 3: VRB induces SG formation in cells and organoids. (A) SG markers TIA-1, G3BP1 and eIF4B (green) were detected in untreated, arsenite-treated (45 min, 0.25 mM) and VRB-treated (1 h, 75 µM) U2OS cells by immunofluorescence. Hoechst 33342 DNA stain is shown in blue. Arrowheads mark some SGs. Scale bar: 10 µm. (B) SG formation was examined in patient-derived breast organoids originating from either healthy tissue or from cancerous tissue. Organoids were treated with arsenite (1 h, 0.25 mM) or VRB (2 h, 75 μM). SGs were detected with anti-G3BP1 antibody (magenta). Phalloidin staining of actin (green) was used to detect the cell outline. Hoechst 33342 DNA stain is shown in blue. Images on the left show 3D presentations of the organoids. Images in the middle show organoid sections, with dashed boxes indicating regions shown in the enlargements on the right. Arrowheads mark SGs. Scale bars: 10 µm. Images in A and B are representative of three experiments.
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