Figure 2. Sestrin2 suppresses mTORC1 and protein synthesis in response to ER stress.

(a-e) At 48 hr after infection with shRNA lentiviruses for luciferase (Con) or Sestrin2, HepG2 cells were treated with PA for 9 hr and analyzed by immunoblotting (n = 3). AMPK signaling activity was monitored by phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). mTORC1 signaling activity was monitored by phosphorylation of p70 ribosomal protein S6 kinase (S6K), ribosomal protein S6 and eIF4E-binding protein (4E-BP). (f-h) 2-month-old WT or Sesn2^−/− mice kept on LFD were injected with tunicamycin (Tm, 500 mg per kg body weight i.p.). After indicated hr, livers were analyzed by immunoblotting (n = 4). (i,j) Relative intracellular ATP levels of indicated cells (i, n = 3) and liver tissues (j, n = 4). As a positive control for ATP depletion, HepG2 cells were treated with oligomycin (OM, 5 μg ml⁻¹) for 1 hr. (k-r) HepG2 cells transduced with sh-Con or sh-Sesn2 (k,m, n = 5) or primary hepatocytes (1°-HPC) isolated from WT or Sesn2^−/− mice (l,n, n = 4) were treated with BSA (−) or PA for 9 hr. 2-month-old WT or Sesn2^−/− mice were injected with Tm (o,q, n = 4) or kept on LFD or HFD for additional 2 months (p,r, n = 4). After indicated treatments, newly synthesized proteins in cells (k-n) and liver tissues (o-r) were visualized and quantified by Click-IT AHA® labeling system. The protein bands denoted by stars correspond to serum albumin (70kD), which is the most actively synthesized protein in normal hepatocytes. All data are shown as the mean ± s.e.m. P values are from Student’s t test. Molecular weight markers are indicated in kDa.