P ampk

Total proteins in placenta tissue or cells were extracted with RIPPA lysis buffer (Beyotime, China) and quantified the concentration by BCA Protein Assay Kit (Beyotime, China). Protein samples were separated using 10% or 12% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, Canada). Membranes were blocked with 5% milk (Boster, China), incubated overnight at 4 °C with primary antibody and further incubated with HRP-labelled corresponding source of secondary antibody (Boster, China) at 37 °C for 1 h. Primary antibodies (Zhongshan, China: ACTB, CST: LC3, #83,506, Atg5, #12,994, Beclin-1, #3495, P62, #5114, mTOR, #2983, p-mTOR, #5536, Akt, #4691, p-Akt, #4060, Raptor, #2280, p-Raptor, #2083, AMPK, #5832, p-AMPK, #2535, Santa: PINK1, sc-517353, Parkin, sc-32282, Phb2, sc-133094) were diluted in 5% milk in PBST. The band intensity was visualized using enhanced chemiluminescence reagent (Bio-Rad, USA) and quantified by Image J software.

The H9c2 cell line was obtained from ATCC (Manassas, VA,USA), sodium hydrogen sulfide (NaHS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dorsomorphin dihydrochloride (HY-13418) was obtained from MCE. Glucose, streptozotocin (STZ), dl-propargylglycine (PAG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM 5.5 mM D-Glucose), penicillin, streptomycin and fetal bovine serum (FBS) were from Hyclone (Grand Island, NY), antibodies against SIRT6, AMPK, p-AMPK, LC3A/B, BECLIN1, ATG5, ATG16L1, P53, P21, P16 were from Boster Biological Technology (Ltd. Wuhan, China. Bicin choninic Acid (BCA) Protein Assay kit, Enhanced Chemiluminescence Reagent kit, SDS-PAGE Gel Preparation kit and phenyl methyl sulfonyl fluoride (PMSF) were purchased from Beyotime Institute of Biotechnology(Shanghai, China), SPiDER-βGal was purchased from dojindo (Japan).