Figure 3.

ER stress-preconditioned cells do not respond to additional acute ER stress treatment but do respond to acute oxidative stress in a cell type manner

(A) Schematic diagram of stress treatments.

(B) (i) Representative Western blot of the ISR expression profile (PERK-P, PERK, eIF2α-P[S51], pan-eIF2α, CHOP, and GADD34) and global newly synthesized proteins (puromycin incorporation assay) in SH-SY5Y, U373 and MO3.13 cells treated with vehicle (DMSO), acute stress inducers (Tg 1 μM for 1h and SA 125 μM for 30 min) or chronic ER stress (Tg 300 nM for 24h) subsequently challenged with previously described acute stress treatments. (ii) Mean expression levels of eIF2α-P[S51] normalized to total eIF2α levels (top panel) and puromycin-labeled nascent proteins normalized to housekeeping GAPDH levels (bottom panel) upon the previously described stress conditions. Fold-change relative to vehicle-treated cells was calculated and analyzed using one-way ANOVA (mean ± SEM; N = 3–9; ∗p < 0.05, ns: non-significant). Chronic ER stress conditions are highlighted in green.

(C) (i) Representative Western blot of eIF2α-P[S51], pan-eIF2α, and global newly synthesized proteins (puromycin incorporation assay) in SH-SY5Y, U373, and MO3.13 cells treated with ISRIB (200nM) for 1h alone, Tg 300 nM for 24h added with SA 125 μM in the last 30min, or combination of both. DMSO for 24h was used as a vehicle. (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 = 3–4; ∗∗∗∗p ≤ 0.001, ∗∗∗p ≤ 0.001, ∗p < 0.05, ns: non-significant).