Skip to main content
. Author manuscript; available in PMC: 2013 Jun 25.
Published in final edited form as: Nat Cell Biol. 2013 Apr 28;15(5):481–490. doi: 10.1038/ncb2738

Figure 4.

Figure 4

ATF4 and CHOP increase protein synthesis leading to cell death. (a) Increased protein synthesis by ATF4 and CHOP. ATF4 and CHOP were expressed either alone or together in Chop+/+ or Chop–/– MEFs. After 24 h, cells were pulse-labelled with [35S]methionine/cysteine (n = 3 independent experiments; see Methods). (b, c) Ablation of translation attenuation by ATF4- and CHOP-mediated GADD34 induction. After forced expression of ATF4 and CHOP, MEFs were treated with 1 μM thapsigargin (Tg) for an hour and analysed by western blotting (b) and metabolic labelling (n = 3 independent experiments; c). (d) Effect of Gadd34 deletion on ATF4/CHOP-mediated increase in protein synthesis. ATF4 and CHOP were expressed in WT and Gadd34–/– MEFs, followed by metabolic labelling (n = 3 independent experiments). (e, f) Effect of translation attenuation on cell survival. Cells were transfected with scrambled (Scr) or Rpl24 siRNAs, followed by Ad-ATF4 and Ad-CHOP infection. Protein synthesis at 24 h (e) and cell viability at 48 h (f) were measured (n = 3 independent experiments). (g–i) Effect of unrestricted protein synthesis on survival. (g) Translational recovery after one hour Tg (1 μM) treatment in Eif2αA/A and Eif2αS / S (left panel) or Atf4+/+ and Atf4–/– MEFs (right panel; n = 3 independent experiments). (h) MEFs were treated with Tm (2 μg ml–1) for 24 h, and cell viability was measured (n = 3 independent experiments). (i) Cell lysates were collected at indicated times for western blot analysis. (j–l) Effect of GADD34 overexpression on cell survival. (j) Translational recovery after one hour Tg (1 μM) treatment in WT, Eif2αA/A and Atf4–/– MEFs after forced expression of either Gadd34ΔC or Gadd34ΔN (n = 3 independent experiments). (k) Indicated MEFs were treated with Tm (2 μg ml–1) for 24 h and viability was measured (n = 3 independent experiments). (l) Western blot analysis of cell lysates from MEFs treated with Tm (2 μg ml–1) for the indicated times. All error bars represent means ± s.e.m. Uncropped images of blots are shown in Supplementary Fig. S7.