Figure 4.

eIF2Bδ remodeling of small eIF2B bodies is transient during cellular stress and partially dictated by eIF2α-P[S51] in a cell type dependent manner

(A) (i) Confocal images of SH-SY5Y, U373, and MO3.13 expressing eIF2Bε-mGFP and immunolabelled with anti-eIF2Bδ subjected to acute stress inducers (Tg 1 μM for 1h and SA 125 μM for 30min) or chronic ER stress (Tg 300 nM for 24h) subsequently challenged with previously described acute stress treatments. Scale bar: 10 μm. (ii) Mean percentage of eIF2Bε-mGFP-containing small (top panel) and large (bottom panel) bodies co-localizing with eIF2Bδ of a population of 30 cells per biological repeat. Fold-change relative to vehicle-treated cells was calculated and analyzed using one-way ANOVA (mean ± SEM; ∗p < 0.05; ns, non-significant).

(B) (i) Representative Western blots of the ISR expression profile (PERK-P, PERK, eIF2α-P[S51], pan-eIF2α, CHOP, and GADD34), global newly synthesized proteins (puromycin incorporation assay) and loading control GAPDH in SH-SY5Y, U373 and MO3.13 cells treated with vehicle (DMSO), GSK2606414/PERKi (500 nM), Tg (1μM) or co-treated with PERKi and Tg (PERKi + Tg) for 1h. (ii) Confocal images of SH-SY5Y, U373 and MO3.13 cells expressing eIF2Bε-mGFP and immunolabelled with primary anti-eIF2Bδ subjected to previous treatments. Scale bar: 10 μm. (iii) Mean percentage of eIF2Bε-mGFP-containing small (left panel) and large (right panel) bodies co-localizing with eIF2Bδ of a population of 30 cells per biological repeat. Fold-change relative to vehicle-treated cells was calculated and analyzed using one-way ANOVA (mean ± SEM; N = 3; ∗p < 0.05).