obtained from CellzDirect Inc (Austin, TX). The cells were cultured in DMEM/F12 (1:1) culture medium. The human normal hepatocytes used were at least 90% viable before treatment. Anti-caspase 8, anti-caspase 10, anti-caspase 3, anti- cytochrome c, anti-HSP90, anti-Bcl-xL, anti-IKK-b, anti-IkB-a, anti-p-IkB-a, anti-Mcl-1, anti-Bak, anti-DR4, anti-DR5, and anti- Bid primary antibodies were obtained from Cell Signaling Technology(Beverly, MA); Dihydroethidium (DHED), N-acetyl- L-cysteine (NAC), Bis (maleimido) hexane (BMH)/DSS, DMAG- 17, Mito Tracker (Red) CMXRos, IKK inhibitor, NEMO-binding domain peptide (NBD): MAPK inhibitor: PD98059, P38 inhibitor: SB203580, and JNK inhibitor: SP600125 were obtained from Invitrogen (Carlsbad, CA); anti-b-actin, anti-Bax 6A7 monoclonal antibodies, Hoechst 33258, and 49, 69-Diamidino-2-phenylindole (DAPI) were obtained from Sigma (St. Louis, MO); z-IETD-FMK and z-VAD-FMK were obtained from Calbiochem (San Diego, CA). Anti-Bax (N-20) primary polyclonal antibody, goat anti- rabbit horseradish peroxidase (HRP) conjugated secondary antibody, Goat anti-rabbit secondary antibody conjugated with FITC, and protein G plus-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Annexin-V apoptosis detection kit was obtained from BD Bioscience (San Diego, CA). Lexatumumab was kindly provided by Human Genome Science Inc. [12,17]. Hoechst staining assay Apoptosis was determined through nuclear morphology change. After treatment with different stimuli, cells were stained with Hoechst 33258 at 37uC for 10 min. Cellular DNA fragmentation/ nucleus condensation was detected using an Olympus fluorescent microscope. Apoptotic cell death ratio was assessed through calculating the number of apoptotic cells with condensed nuclei in six to eight randomly selected areas. DNA ladder assay A DNA ladder assay was performed as previously described [18] with modification. Briefly, cells treated under different conditions were harvested and washed with 16phosphate buffered saline (PBS). Cell pellets were resuspended with lysis buffer (1% NP-40 in 20 mM EDTA, 50 mM Tris-HCl, pH 7.5). The samples were centrifuged at 1,6006g for 5 min and supernatants were transferred to new tubes. SDS (final concentration 1%) and RNase A (final concentration 5 mg/ml) were added to the supernatants, and the mixture was incubated at 56uC for 2 h. After addition of proteinase K (final concentration 2.5 mg/ml), the samples were incubated at 37uC for 2 h. DNA was precipitated with the addition of 10 M ammonium acetate and ethanol, washed once with 70% ethanol and then dissolved in water and separated by electrophoresis in 1% agarose gel. Fluorescence assisted cell sorting (FACS) assay Apoptosis was measured using the Annexin-V detection kit according to the manufacturer’s instructions. Flow cytometric analysis was performed to monitor the green fluorescence of the FITC-conjugated Annexin-V and the red fluorescence of DNA- bound propidium iodide (PI). All data were analyzed with a Cell Quest software (BD). Western blotting analysis Cells were harvested and washed twice with 16PBS. The cell pellets were resuspended in lysis buffer containing Nonidet P-40 (10 mM HEPES, pH 7.4, 2 mM EGTA, 0.5% Nonidet P-40, 1 mM NaF, 1 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride, CHX and Lexa Combination-Induced Apoptosis in HCC 1 mM dithiothreitol, 50 mg/ml trypsin inhibitor, 10 mg/ml aprotinin, and leupeptin) and incubated on ice for 30 min. After centrifugation at 12,0006g at 4uC for 15 min, the supernatant was transferred to a new tube and the protein concentration was determined. Equivalent samples (20 mg of protein) were subjected to SDS-PAGE on 12% gels. The proteins were transferred to nitrocellulose membranes and probed with the indicated primary antibodies, followed by the appropriate secondary antibodies conjugated with horseradish peroxidase. Immunoreactive bands were detected using enhanced chemiluminescence (ECL) (Pierce, Rockford, IL). The molecular sizes of the proteins detected were determined by comparison with prestained protein markers (Bio- Rad, Hercules, CA). Immuno-fluoresence staining Immunostaining was performed as described previously [19] with modification. Briefly, cells seeded on cover-slips in culture dishes were treated with Lexa in the presence of CHX for 6 h. Mito-Tracker (Red) was directly added to cultures and incubated for 15 min. Cells were washed with 16 PBS, fixed with 3.7% paraformaldehyde (PFA) at 37uC for 15 min, and then quenched with 50 mM NH4Cl. After washing with PBS twice, cells were permeabilized with 0.2% Triton X-100 in PBS, then blocked with PBS containing 5% goat serum for 30 min. Cells were further incubated with primary antibody cytochrome c overnight at 4uC, followed by staining with FITC conjugated goat anti-rabbit IgG (1:200) for 2 h at room temperature. The cover-slips were then extensively washed and mounted in Mowiol with DAPI dye. Fluorescent images were obtained using an Olympus fluorescent microscope. Bax cross-linking In vitro protein cross-linking was carried out as previously described [20] with minor modification. Cells treated with different conditions were harvested. Cell lysates were prepared after lysis with HEPES-CHAPS lysis buffer (20 mM HEPES pH 7.2, 150 mM NaCl, 2 mM EDTA, 10 mM glucose, 2% CHAPS). Bis (maleimido) hexane (BMH) substrate or dimethyl sulfoxide (DMSO) was added at a final concentration of 2 mM. The samples were incubated at room temperature for 30 min and quenched by the addition of 1 M Tris-HCl (pH 7.5). An in vivo protein cross linking assay was performed as previously described. Following treatments with Lexa or Lexa combined with CHX, cells were washed with conjugating buffer containing 150 mM NaCl, 20 mM HEPES (pH 7.2), 1.5 mM MgCl2, and 10 mM glucose. DSS in DMSO was added for a final concentration of 2 mM. After reaction at room temperature for 30 min, the cross- linker was quenched by the addition of 1 M Tris-HCl (pH 7.5) for a final concentration of 20 mM. Samples were then solubilized in 1% NP-40 and centrifuged at 12,0006g for 10 min. A 20-ml aliquot from the 400 ml cross-linked lysate was analyzed by Western blotting with Bax (N-20) polyclonal antibody. ROS detection The intracellular production of ROS was measured by fluorogenic probe dihydroethidium-(DHED) based fluorescent staining. Briefly, after various treatments, cells were incubated with DHED (5 mM) for 30 min at 37uC, then rapidly rinsed twice with 16 PBS and observed under fluorescent microscope. siRNA knockdown siRNA knockdown was performed as previously described [21]. Smart-pool pre-designed siRNA duplexes targeted against human PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e16966