Enhanced chemiluminescent detection kit

To determine whether autophagy-related molecules are regulated by the NF-κB pathway after SN treatment, J5 cells were treated with 100, 250 or 500 µM of SN for 24 h. Total protein was collected and prepared from cells. Cytosolic p-IκB, IκB, and NF-κB as well as nuclear NF-κB (p65) expression were determined using SDS-PAGE and immunoblot assay (28, 29) . Bands were visualized using hydrogen peroxide/ tetrahydrochloride diaminobenzidine or an enhanced chemiluminescent detection kit (Amersham Life Science) and were quantitated with an AlphaImager 2000 (Alpha Innotech).
For the NF-κB-DNA binding activity assay, nuclear extracts were obtained from cell pellets using an NE-PER extraction kit (Thermo Scientific) as per the manufacturer's instructions. NF-κB-DNA binding activity within the nuclear fraction was determined using an NF-κB (p65) transcription factor activity assay kit (Cayman Chemical Co., Ann Arbor, MI, USA) as per the manufacturer's instructions.
Statistical analysis. Statistical analyses were performed using SAS software (SAS Institute). Analysis of variance (ANOvA) and Duncan's multiple-range test were used to identify significant differences among the means (p<0.05).

To elucidate the signal transduction pathways by which autophagy was induced by SN in J5 cells, we analyzed the protein expression of autophagy regulators of pre-autophagosome and autophagosome formation. J5 cells were treated with 100, 250 or 500 µM of SN for 24 h. Total protein was collected and prepared from cells. Nuclear extracts were obtained from cell pellets using an NE-PER extraction kit (Thermo Scientific, Rockford, IL, USA) as per the manufacturer's instructions.
Cellular protein concentration of cells was assayed by the method of Lowry et al (27) . Cytoplasmic PI3K-I, PI3K-III, Akt, mTOR, Beclin-1, LC3-II, p53, DRAM and TIGAR as well as nuclear p53 expression were determined using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot assay (28, 29) . Bands were visualized using hydrogen peroxide/tetrahydrochloride diaminobenzidine or an enhanced chemiluminescent detection kit (Amersham Life Science, Buckinghamshire, UK) and were quantitated with an AlphaImager 2000 (Alpha Innotech, San Leandro, CA, USA).

Proteins were prepared as described previously [15] . Freeze-clamped LV tissues (200 -300 mg) were homogenized briefly in 10 volumes of lysis buffer containing (in mM) 20 Tris-HCl (pH, 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na4P2O7, 1 Na3VO4, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma-Aldrich, Missouri, USA), 1% (v/v) Triton X-100 and 10% (v/v) glycerol. The homogenates were centrifuged twice at 20,000 g at 4°C for 15 min, and the supernatants were saved as total proteins. Protein concentrations were determined by the BCA method. Western blot analysis of BNP was performed by loading 20ug of total protein per well on an SDS-PAGE gel. Proteins were transferred to a PVDF membrane (Bio-Rad, California, USA), blocked with 5% non-fat dry milk and probed with specific anti-brain natriuretic protein (BNP) (Ab19645, Abcam, USA) antibody at dilution of 1:1000 and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ab8245, Abcam, USA) at dilution of 1:5000. The immunoreaction was visualized using HRP-conjugated goat anti-rabbit IgG secondary antibody (sc-2357, Santa Cruz Biotechnology, USA) and enhanced chemiluminescent detection kit (Amersham, London, UK), exposed to X-ray film, and quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad, California, USA).