Annexin V and propidium iodide labeling B16F10-Nex2 cells (56105 cells/well) were cultured in 6-well plates and further incubated with 20 or 50 mM jacaranone or complete medium (control) for 24 h at 37uC. Treated and untreated cells (1.06106) were washed three times with PBS and harvested with a cell scraper. Apoptotic/necrotic cells were detected using the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, MO). Cells were incubated with binding buffer (10 mM HEPES/NaOH, pH 7.5, 140 mM NaCl and 2.5 mM CaCl2) in presence of propidium iodide (PI) and FITC- labeled annexin V (AV) for 10 min at room temperature and analyzed by flow cytometry (BD Bioscience FACSCanto II equipment, Franklin Lakes, NJ), using FlowJo software (TreeStar Inc., Ashland, OR). DNA degradation B16F10-Nex2 cells (56105/well) cells were seeded on 6-well plates and incubated for 12 h at 37uC. Cells were then treated with 100 mM jacaranone or left untreated, and incubated at 37uC for 18 h. DNA was isolated after cells were lysed in TELT buffer (50 mM Tris–HCl pH 8.0, Triton X-100 0.4%, 2.5 mM EDTA pH 9.0, and 2.5 M LiCl). The lysate was centrifuged for 20 min (12,000 g) at 4uC. Buffer-equilibrated phenol was added (1:1, v/v), followed by centrifugation and addition of chloroform (1:1, v/v) to the resulting aqueous phase. Tubes were centrifuged (15 min, 12,000 g, 4uC), the aqueous phase was collected and DNA was precipitated with sodium acetate 3 M, pH 7.0 and absolute ethanol (1:0.1:2.5, v/v/v) after incubation at – 80uC for 20 min. Precipitated DNA was pelleted and diluted in 50 mg/ml of RNAse-A (Invitrogen, Carlsbad, CA). The extracted DNA was subjected to electrophoresis on 1% Agarose gel with ethidium bromide (0.5 mg/ml) in TBE buffer (2 mM EDTA, 90 mMT ris– HCl, 90 mM boric acid, pH 8.0) at 100 V. One thousand base- pair (1 kb) ladder molecular weight markers (Gibco, Grand Island, NY) were used. After electrophoresis, DNA was photographed with a digital camera (Kodak, EDAS DC290) under ultraviolet (UV) illumination. TUNEL Assay Apoptotic cell death was also assessed by terminal deoxynu- cleotidyltransferase dUTP nick end-labeling (TUNEL) assay kit (In Situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, Indianopolis, IN), according to manufacturer’s instruc- tions. Briefly, 1.06104 B16F10-Nex2 cells were seeded onto round glass coverslips and cultivated for 24 h before incubation with 50 mM jacaranone for 3 h. After treatment, cells were washed with PBS and fixed with 2% formaldehyde for 30 minutes at room temperature. Next, cells were permeabilized with 0.1% Triton X- 100 for 30 min at room temperature and incubated with TdT enzyme (terminal deoxynucleotidyltransferase) in the reaction buffer with dUTP-fluorescein at 37uC for 1 h, followed by staining with 10 mg/mL of 49,6-diamidino-2-phenylindole (DAPI, Invitro- gen, Carlsbad, CA) for 10 min in the dark. Apoptotic cells were visualized on Olympus BX-51 fluorescence microscope using 60X oil immersion objective. Images were processed with ImageJ (http://rsb.info.nih.gov/ij/). As a positive control, cells were treated with 1 mg/mL actinomycin D (Sigma-Aldrich, St. Louis, MO) for 1 h. Detection of caspase activity by colorimetric assay For evaluation of caspase activity, B16F10-Nex2 cells (1.06106) were cultured and treated with or without 20 mM jacaranone. After the indicated periods, cells were harvested and tested for Antitumor Activity of Jacaranone caspase activity using the ApoTarget Caspase Colorimetric Protease Assay Sampler Kit (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol. Briefly, the cells were resuspended in 50 mL of chilled Cell Lysis Buffer for 10 min on ice bath. The lysates were centrifuged at 10,000 g for 1 min, and 200 mg of the protein, determined by Bradford’s method [10] was incubated with 200 mM substrate VDVAD-pNA (caspase-2), DEVD-pNA (caspase-3), IETD-pNA (caspase-8), and LEHD-pNA (caspase-9) at 37uC for 2 h in a 96-well plate. Absorbance of pNA was read at 405 nm in a microplate reader (SpectraMax-M2, Software Pro5.4, Molecular Devices, Sunnyvale, CA). Chromatin condensation analysis Murine melanoma cells (1.06104) were cultivated overnight on round glass coverslips and incubated with or without 20 or 50 mM jacaranone for 3 h. Then, cells were washed with PBS, fixed for 30 min at room temperature with 2% formaldehyde, and stained with 2 mM Hoechst 33342 (Invitrogen, Carlsbad, CA) for 10 min to study the state of chromatin. Cells were visualized on an Olympus BX-51 fluorescence microscope with immersion oil at 60X magnification. Apoptotic cells were detected by condensed or fragmented nuclei. Apoptotic morphological changes by transmission electron microscopy Transmission electron microscopy (TEM) was used to provide ultrastructural evidence of jacaranone-induced apoptosis. B16F10- Nex2 cells (56104) were seeded on 12-mm round aclar plastic coverslips housed in 6-well plates, and incubated with 50 mM jacaranone for 3 h at 37uC. After fixation with 2.5% glutaralde- hyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 at room temperature for 20 h, cells were washed in the same buffer for 10 min, and then post-fixed with 1% osmium tetroxide in 0.1 M cacodylate at pH 7.2 for 30 minutes. Next, cells were washed with distilled water for 10 minutes at room temperature, treated with an aqueous solution of 0.4% uranyl acetate for 30 minutes and washed again with distilled water for 10 minutes. After fixation, cells were dehydrated in a graded series of ethanol (70%, 90% and 100%) and propylene oxide washes, and embedded in Epon. Semi-thin sections were obtained from selected areas, collected on copper grids, stained with 1% uranyl acetate and lead citrate, and examined on a Jeol 1200 EXII electron microscope (Tokyo, Japan). Detection of mitochondrial membrane potential (Dym) The cationic lipophilic dye tetramethylrhodamine ethyl ester (TMRE) enters the cell in the form of an ester that is subsequently hydrolyzed and converted to tetramethylrhodamine, which is reversibly accumulated in the negatively charged mitochondrial matrix depending on mitochondrial membrane potential. To analyze the Dym after jacaranone treatment, 1.06105 B16F10-Nex2 cells were grown for 24 h in a 12-well culture plate and incubated with 50 mM jacaranone for the indicated times at 37uC. Cells were gently washed with PBS and loaded with 20 nM TMRE (Molecular Probes, OR, USA) for 10 min at 37uC. Cells were detached with PBS/Trypsin/EDTA and the uorescence was measured on FACSCanto II (BD Bioscience, Franklin Lakes, NJ), using FACSDiva software (BD Bioscience, Franklin, NJ) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38698