Journal: iScience
Article Title: Chronic stress antagonizes formation of stress granules
doi: 10.1016/j.isci.2025.114556
Figure Lengend Snippet: Chronic stress slows down translation at the elongation step (A) Polysome profiles from U2OS cells. NT, black; 2 μM harringtonine incubation for 3 min, red; 5 min, blue; 8 min, green. For these treatments cell without (left) or with (right) 24 h of SA 10 μM pre-incubation were used. Two biological replicates (individual experiments) were done. The polysome/monosome ratio was calculated and shown on the right. The same colors of the circle dots in the graph are from the same independent experiment. (B) Polysome profiles from RNase A (0.5 mg/mL)-digested lysates of U2OS cells. NT, black; 1 μg/mL ANS, pink; 10 μM SA incubation for 24 h, blue; 100 μM SA treatment for 1 h with 10 μM SA pre-incubation for 24 h, green. The polysome/monosome ratio was calculated and shown on the right. (C) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, or 1 μg/mL ANS for 15 min. Cell lysates were subjected to western blotting using antibodies for p -eEF2, total eEF2, and β-actin. (D) U2OS cells were subjected to treatment with 10 μM SA, or 12.5 μM NFV (nelfinavir) for 24 h. Cell lysates were subjected to western blotting using antibodies for p -eEF2, total eEF2, and α-tubulin. (E) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 12.5 μM NFV for 24 h. Cells were examined for the presence of the core SG markers G3BP1 (green), eIF3b (red), and DAPI (blue). Scale bars represent 20 μm. G3BP1- and eIF3b-positive cells were quantified (right graph). p values were assessed using a one-way ANOVA ( p ∗∗∗∗ <0.0001). Results are mean ± S.E.M. ( n = 3). (F) U2OS cells were subjected to treatment with 10 μM SA for 1, 4, 12, and 24 h or 1 μg/mL ANS for 15 min. Cell lysates were subjected to western blotting using antibodies for p -eEF2, total eEF2, and β-actin. (G) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA for 0, 1, 4, 12, and 24 h. Cells were examined for the presence of the core SG markers G3BP1 (green), eIF4G (red), and eIF3b (blue). Scale bars represent 20 μm. All positive cells were quantified. p values were assessed using a one-way ANOVA (vs. 1 h pre-incubation of SA +1 h SA; p ∗∗∗∗ <0.0001). Results are mean ± S.E.M. ( n = 3). (E and G) All experiments were done for each of the three biological replicates (independent experiments).
Article Snippet: For any experiments, cells were grown to ∼70% confluency and then treated as indicated in figure legends: sodium arsenite (SA, Sigma), rocaglamide A (RocA, MedChemExpress), menadione (Mena, Sigma), doxorubicin (Drb, AdooQ Bioscience), thapsigargin (Tg, Invitrogen), tunicamycin (Tun, Sigma), brefeldin A (BA, Sigma), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma), 2-deoxy-D-glucose (2DG, Sigma), nelfinavir (NFV, MedChemExpress).
Techniques: Incubation, Western Blot
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