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

(a-c) HepG2 cells stably transduced with Sestrin2 shRNA (sh-Sesn2) were treated with PA for indicated hr (a) or 6 hr (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 hr 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 hr, cells were treated with BSA (−) or PA for 6 hr and analyzed by immunoblotting (n = 3). (f,g) At 48 hr after infection with shRNA lentiviruses for luciferase (Con) or TSC2 (sh-TSC2), HepG2 cells were treated with BSA (−) or PA for 6 hr and analyzed by immunoblotting (n = 3). (h-k) 5-month-old Sesn2^−/− mice kept on HFD for 3 months were transduced once with adenoviruses expressing GFP (Con, n = 5) or constitutive active AMPK (AMPK^CA, 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.