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. 2024 Aug 30;27(9):110851. doi: 10.1016/j.isci.2024.110851

Figure 5.

Figure 5

ISRIB restores translation during chronic ER stress while increasing the eIF2Bδ composition of small eIF2B bodies predominantly in astrocytic cells

(A) (i) Confocal images of SH-SY5Y, U373, and MO3.13 expressing eIF2Bε-mGFP and immunolabelled with primary anti-eIF2Bδ subjected to ISRIB (200nM) alone 1h or in combination with preconditioned chronic ER stress treatment (Tg 300nM 24h + ISRIB last 1h). Scale bar: 10 μm. (ii) Mean percentage of eIF2Bε-mGFP-containing small (top panel) and large (bottom panel) bodies co-localizing with eIF2Bδ. Fold-change relative to vehicle-treated cells was calculated and analyzed using one-way ANOVA (mean ± SEM; N = 3; ∗∗p ≤ 0.01, ∗p < 0.05).

(B) (i) Western blotting of global newly synthesized proteins (puromycin incorporation assay) and loading control GAPDH in SH-SY5Y, U373, and MO3.13 cells treated with the same conditions as described previously. (ii) Mean expression levels of puromycin-labeled nascent proteins normalized to housekeeping GAPDH levels. Fold-change relative to vehicle-treated cells was calculated and analyzed using one-way ANOVA (mean ± SEM; N = 5–9; ∗∗∗∗p ≤ 0.001, ∗∗∗p ≤ 0.001, ∗p < 0.05).