Western blot analysis

KKU-213L5 cells were incubated with honokiol at indicated concentrations for 20 h. For the analysis of intracellular proteins, treated cells were washed with ice-cold PBS before cell lysis using RIPA lysis buffer plus protease inhibitor cocktail (AMRESCO, OH, United States). Then, protein lysates were collected by centrifugation and the total protein concentration was qualified by using Bradford assay. In addition, the secreted protein was collected from conditioned medium, which was concentrated using Amicon^® Ultra-2 Centrifugal Filter units (Millipore, MA, United States) following the manufacturer’s instruction for 20× final concentration. The protein was then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto the polyvinylidene fluoride membranes. After that, the non-specific binding was blocked with 5% skim milk buffer for 1 h before washing with TBST buffer. The membranes were then incubated with each primary antibody, anti-caspase 3 (Cell Signaling, MA, United States), anti-HMGB1 (ELabScience, TX, United States) and anti-HSP90 (Merck, Darmstadt, Germany) antibodies with gentle shaking at 4 °C overnight. Then, membranes were washed with TBST and incubated with horseradish peroxidase-linked anti-rabbit antibody (Cell Signaling, MA, United States) for 1 h at room temperature, and washed again before incubated with detection reagent. The image was developed by Chimidoc™ XRS (Bio-rad, CA, United States) and analyzed by Image Lab (Bio-rad, CA, United States).