Mitochondrial Isolation Mitochondria were isolated from untreated Saos-2 and U-2 OS cells. These cells were washed two times with cold PBS, resuspended in hypotonic buffer (1 mM EDTA, 5 mM Tris–HCl, 210 mM mannitol, 70 mM sucrose, 10 mM Leu-pep, 10 mM Pep-A, and 100 mM PMSF), manually homogenized, and subsequently centrifuged at 6006 g for 5 minutes at 4uC. The supernatant was centrifuged at 15,0006 g for 15 minutes at 4uC. The resulting cytosolic supernatant was discarded and the mitochondrial pellet was resuspended in cold reaction buffer (2.5 mM malate, 10 mM succinate, 10 mM Leu-pep, 10 mM Pep-A, and 100 mM PMSF in PBS). Amplex Red Assay Generation of reactive oxygen species (ROS) was quantified with Amplex Red (Molecular Probes, Eugene, OR, USA). Opaque 96-well plates were equally loaded with isolated mitochondria suspended in cold reaction buffer, with 20 mg of protein/well. The Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA USA) was used for protein quantification as per manufacturer’s protocol. Isolated mitochondria were incubated with the indicated concen- trations of drugs, Amplex Red reagent at a final concentration of 50 mM, and horseradish peroxidase (Sigma-Aldrich Canada, Mississauga, ON, Canada) in the ratio of 6 U/200 mL. Paraquat (PQ) (Sigma-Aldrich Canada, Mississauga, ON, Canada) was used as a positive control at 250 mM. Fluorescence readings were acquired after 2 hours of incubation at Ex. 560 nm and Em. 590 nm on a spectrofluorometer (SpectraMax Gemini XS, Molecular Devices, Sunnyvale, CA, USA). Fluorescence readings were expressed as relative fluorescence units (RFU). Treatment of Isolated Mitochondria & Evaluation of Apoptogenic Factor Release Mitochondria isolated from Saos-2 and U-2 OS cells were directly treated with JCTH-4 and CC at the indicated concentra- tions for 2 hours in cold reaction buffer (2.5 mM malate, 10 mM succinate, 10 mM Leu-pep, 10 mM Pep-A, and 100 mM PMSF in PBS). The control group was treated with solvent (Me2SO). After treatment, mitochondrial samples were vortexed and centrifuged at 15,0006 g for 15 minutes at 4uC. The resultant supernatant and mitochondrial pellet (resuspended in cold reaction buffer) samples were subjected to western blot analyses to monitor for mitochon- drial release or retention of apoptogenic factors. PST Analogue & Curcumin for Osteosarcoma Therapy Cellular Lysate Preparation Cells were treated for 72 hours with the indicated concentra- tions of JCTH-4, CC, and TAM, and subsequently homogenized manually in cold hypotonic buffer (10 mM Tris HCl at pH 7.2, 5 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1% Triton-X-100; 10 mM Leu-pep, 10 mM Pep-A, and 100 mM PMSF). Cell lysates were stored at -20uC until use. Western Blot Analyses SDS-PAGE was performed on the protein samples. Electropho- resed proteins were transferred to a nitrocellulose membrane. For 1 hour, membranes were blocked with a 5% w/v milk TBST (Tris- Buffered Saline with Tween-20) solution. The membranes were probed with various primary antibodies overnight at 4uC for microtubule-associated protein1 light chain 3 (LC3) raised in rabbit (1:500) (Novus Biologicals, Cat. No. NB100-2220, Littleton, CO, USA), b-Actin raised in mouse (1:1000) (Santa Cruz Biotechnology, Inc., Cat. No. sc-81178, Paso Robles, CA, USA), apoptosis inducing factor (AIF) raised in rabbit (1:1000) (Abcam, Cat. No. ab1998, Cambridge, MA, USA), endonuclease G (EndoG) raised in rabbit (1:1000) (Abcam, Cat. No. ab9647, Cambridge, MA, USA), and succinate dehydrogenase subunit A (SDHA) raised in mouse (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-59687, Paso Robles, CA, USA). Following primary antibody incubation, membranes were washed once for 15 minutes and twice for 5 minutes with TBST and were incubated with an anti-mouse (1:2000) or an anti-rabbit (1:2000) horseradish peroxidase-conjugated secondary antibody (Abcam, Cat. No. ab6728 & ab6802, Cambridge, MA, USA) for 1 hour at 25uC. After secondary antibody incubation, the membranes were washed three times for 5 minutes in TBST. Bands were visualized with enhanced chemiluminescence reagent (Sigma- Aldrich Canada, Cat. No. CPS160, Mississauga, ON, Canada) and densitometry analyses were performed using ImageJ software. Monodansylcadaverine (MDC) Staining MDC (Sigma-Aldrich Canada, Mississauga, ON, Canada) was used to detect autophagic vacuoles. Cells were grown on coverslips and treated with TAM, JCTH-4, and CC at the indicated concentrations and for the indicated durations. After treatment, cells were incubated with 0.1 mM MDC for 15 minutes. Using a Leica DM IRB inverted fluorescence microscope (Wetzlar, Germany), micrographs were taken at 4006 magnification. Figure 1. Comparison of chemical structures. Structures of (A) Pancratistatin (PST) and (B) JC-TH-acetate-4 (JCTH-4). doi:10.1371/journal.pone.0028780.g001 PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28780