Readout was performed using a spectrophotometer

by flow cytometry with 2′,7′-dichlorodihydrofluorescein diacetate and mitochondrial superoxide indicator MitoSOX Red as probes. Briefly, Panc-1 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667251 cells treated with SSHE were incubated with 10 M of H2DCFDA or 5 M of MitoSOX Red in serum-free DMEM for 10 min. The medium was removed, and the cells were detached with a brief treatment of 0.25% trypsin in Hank’s balanced salt solution. After addition of fresh culture medium, the cells were collected by centrifugation, washed once with DPBS and suspended in DPBS. The fluorescence was monitored using the FACSCalibur flow cytometer or under a laser confocal microscope. Western blotting Cell extracts were prepared by lysing cells with RIPA buffer on ice for 20 min. The lysates were centrifuged at 12,000 g for 10 min at 4C,and the supernatants were used for subsequent analyses. SDS–polyacrylamide gel electrophoresis and immunoblot analyses were performed as described previously. The primary antibodies PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19668191 used were rabbit monoclonal anti-SQSTM1/ p62, rabbit polyclonal antiLC3B, rabbit polyclonal anti-phospho-mTOR , rabbit monoclonal anti-mTOR, rabbit monoclonal anti-phospho-AMPK , and rabbit monoclonal anti-AMPK antibody. The secondary antibodies were HRP-conjugated rabbit or anti-mouse IgG. For loading controls, anti–actin antibody was used. Signals were visualized using ECL plus. The membranes were scanned with a Luminoimaging Analyzer LAS4000. Immunofluorescent staining Panc-1 cells treated with solvent alone or SSHE were fixed with 4% formaldehyde/5% sucrose in DPBS for 20 min, rinsed with DPBS, and permeabilized with 0.5% Triton X-100 in DPBS for 4 min. In some experiments, cells were stained with 100 nM of MitoTracker Red CMXRos for 10 min before fixation. The cells were blocked with 3% BSA/0.1% glycine in DPBS for 1 h, rinsed, and then incubated with rabbit monoclonal anti-AIF or rabbit polyclonal anti-LC3B antibody for 1 h. After extensive washing with DPBS, the cells were incubated with Alexa Fluor 488-conjugated goat 4 / 22 Antitumor Activity of Ginger Extract against Pancreatic Cancer anti-rabbit IgG for 1 h. The cells were counterstained with DAPI and observed under a laser scanning confocal microscope. Transmission electron microscopy analysis Untreated Panc-1 cells and the cells treated with 200 g/ml SSHE for 28 h were placed in 2% paraformaldehyde and 2% glutaraldehyde in 30 mM HEPES buffer for 2 h at 4C and post-fixed with 1% OsO4 for 1 h at 4C. The samples were dehydrated with graded alcohol and embedded in TAAB812 resin. Ultrathin K 858 site sections were stained for 1 h in 3% aqueous uranyl acetate, washed, and counterstained with 0.3% lead citrate, and they were examined on a transmission electron microscope. GFP-LC3 plasmid and transfection EGFP-LC3B fusion plasmid was constructed by cloning LC3B cDNA, which was amplified by PCR, into a pCMX-SAH/Y145F-GFP vector. The construct was verified by DNA sequencing. Panc-1 ells were transiently transfected with the plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. After 24 h, the cells were exposed to SSHE and examined under a laser scanning confocal microscope. Animal experiments All animal experiments were performed in compliance with the institutional guidelines for the care and use of animal research. The protocol was approved by the IZUMO Campus Animal Care and Use Committee of Shimane University. All the mice were housed in the animal center of Shimane University Faculty of Medicine under specific pathogen-free c