Figure 1

PERK-dependent translational repression dynamics. (A,B) Polysome profiling demonstrated overall translational repression and partial recovery upon ER stress in PERK WT MEFs but not in PERK −/− MEFs. RNAs were separated on a sucrose gradient (10–50%) using an ultracentrifuge, and the gradients were read using a UV reader. The graph shows the amount of RNA bound by different size polysomes in the different conditions. Polysome/monosme ratios (P/M), used to measure the level of overall translation, as the ratio between the polysome area under the curve (4-somes and up) to the monosome area under the curve, were calculated throughout Tg treatment in PERK WT and PERK −/− cells. (A) PERK WT P/M decreased sharply following Tg treatment and showed relaxation in a time-dependent manner: Control P/M = 5.97; Tg 1 h P/M = 1.17; Tg 2 h P/M: 1.79; Tg 5 h P/M = 2.85; Tg 8 h P/M = 2.38. (B) PERK −/− P/M did not show an immediate decrease following Tg treatment, with the exception of a slight repression after 8 h: Control P/M = 6.35; Tg 1 h P/M = 6.44; Tg 2 h P/M = 6.55; Tg 5 h = 5.72; Tg 8 h = 4.22. (C–H) Selected ER stress response genes altered during Tg treatment in PERK WT and PERK −/− cells. All bar plots depict gene expression TPM values in PERK WT and PERK −/− cells following Tg treatments (Control, Tg 1 h, Tg 2 h, Tg 5 h, Tg 8 h). ER-stress related genes showed three distinct activation patterns: while PDIA4 and DNAJC3 were activated in an PERK-independent manner (C,D); XBP1 and HSPA5 (BiP) have shown a partial dependence on PERK (E,F); HERPUD1 and DDIT3 (CHOP) showed a complete dependence on PERK (G,H).