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

From: Hepatoprotective role of Sestrin2 against chronic ER stress

Figure 2

(ae) At 48 h after infection with shRNA lentiviruses for luciferase (Con) or Sestrin2, HepG2 cells were treated with PA for 9 h and analysed by immunoblotting (n=3). AMPK signalling activity was monitored by phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). mTORC1 signalling activity was monitored by phosphorylation of p70 ribosomal S6K, ribosomal protein S6 and eIF4E-binding protein (4E-BP). (fh) Two-month-old WT or Sesn2−/− mice kept on LFD were injected with Tm (500 mg per kg body weight i.p.). After indicated hours, livers were analysed 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 h. (kr) 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 h. Two-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 (kn) and liver tissues (or) were visualized and quantified using the Click-IT AHA labelling system. The protein bands denoted by stars correspond to serum albumin (70 kD), 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.