Beclin 1

Whole-cell protein lysates were solubilized in 100-200 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) supplemented with a 1% protease inhibitor cocktail (Beyotime, China) for 20 min on ice. The protein concentration was determined using a BCA protein assay kit (Beyotime, China). The proteins (30 μg) were denatured and separated on an 8%-15% discontinuous SDS-polyacrylamide gel (SDS-PAGE) by electrophoresis and subsequently electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature, the PVDF membrane was incubated overnight at 4 °C with the appropriate primary antibody for TLR7 (1:100; Novusbio, USA), MyD88 (1:200; Boster, China), IRF7 (1:1000; Abcam, USA), beclin 1 (1:200; Boster, China), SQSTM1 (1:200; Boster, China), EV71/CA16-VP1 (1:1000; Millipore, USA) or GAPDH (as a loading control, 1:10000; Abmart, China). Then, the membrane was extensively washed three times with TBST prior to incubation for 1 h at room temperature with the corresponding secondary antibody (Abmart, USA) at a dilution of 1:12,000. Finally, the membrane was again extensively washed three times with TBST and then visualized using enhanced chemiluminescence reagents (Beyotime, China) and X-ray films (Kodak, Japan).

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).

Tetrahydrocurcumin was purchased from Yuanye Biotechnology Co. Ltd (Shanghai,China). E-cadherin, ZEB, Snail, Twist, HIF-1α, p-mTOR, mTOR, p-ERK, ERK, GAPDH and β-actin antibodies were purchased from Santa cruz Biotechnology (Santa cruz, CA, USA). Vimentin, N-cadherin, VEGF, MMP-2, MMP-9, LaminA/C, Atg5, Atg7 and Beclin-1 antibodies were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). LY294002, SB203580, Rapamycin, β-catenin, p-Akt, Akt, p-JNK, JNK, p-p38, p38, IκB-α, p65, Hsp90 and GSK-3β antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). Antibody against LC3B was purchased from Abcam (Cambridge, UK). All other reagents were from common commercial sources.