AML12 cells, a non-tumorigenic mouse hepatocyte cell line, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 containing 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, 40 ng/mL dexamethasone and 10% FBS at standard cell culture conditions (5% CO2, 95% air). Negative small interfering RNA (siRNA) or siRNA targeting at Nrf2 and Sirt6 (100 nmol/L; RiboBio, Guangzhou, China) were transiently transfected using lipofectamine 2000 siRNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany). Cells were treated with doxorubicin (Dox, 125–500 nmol/L) or APAP solution (5 mmol/L) at 24 h after siRNA transfection and incubated for 48 h.
Small interfering RNA (siRNA) was transfected to decrease Sirt6 level in AML12 cells. The knockdown efficiency of Sirt6 siRNA was validated by investigating protein expression of SIRT6 using Western blot analysis (Supporting InformationFig. S1 ). Three different siRNA sequences of Sirt6 were compared and the highest one with knockdown efficiency >80% (sequence: GTGCATGTTTCGTATAAGT) was chosen in the current study.
Small interfering RNA (siRNA) was transfected to decrease Sirt6 level in AML12 cells. The knockdown efficiency of Sirt6 siRNA was validated by investigating protein expression of SIRT6 using Western blot analysis (Supporting Information