Figure 4 : Sestrin2 controls ER homeostasis through the AMPK-mTORC1 axis.

From: Hepatoprotective role of Sestrin2 against chronic ER stress

Figure 4

(ac) HepG2 cells stably transduced with Sestrin2 shRNA (sh-Sesn2) were treated with PA for indicated hours (a) or 6 h (b). PBS (Con), AICAR (1 mM), Rapamycin (Rap, 100 nM), PP242 (1 μM), cycloheximide (CHX, 180 μM), BHA (100 μM) and NAC (10 mM) were applied 1 h before treating with PA. AICAR is an AMPK activator, PP242 is an mTOR inhibitor and CHX is a protein translation inhibitor. Protein phosphorylation and expression were examined (a,b) and quantified (c) (n=3). P values were calculated between PA+PBS (Con) and indicated groups. (d,e) Sestrin2-silenced HepG2 cells were transduced with shRNA lentiviruses for luciferase (Con) or Raptor (sh-Raptor). After 48 h, cells were treated with BSA (−) or PA for 6 h and analysed by immunoblotting (n=3). (f,g) At 48 h after infection with shRNA lentiviruses for luciferase (Con) or TSC2 (sh-TSC2), HepG2 cells were treated with BSA (−) or PA for 6 h and analysed by immunoblotting (n=3). (hk) Five-month-old Sesn2−/− mice kept on HFD for 3 months were transduced once with adenoviruses expressing GFP (Con, n=5) or constitutive active AMPK (AMPKCA, n=6) (h,i) or were injected daily with vehicle (PBS, n=6) or AICAR (250 mg per kg body weight per day i.p., n=5; j,k). After 10 days, livers were harvested, and protein phosphorylation and expression were examined (h,j) and quantified (i,k). 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.