(wild type [WT]), SHP−/−
(SKO), SHP nontransgenic control (NC), and hepatocyte-specific SHP transgenic (STG) mice have been described previously (46
). Primary mouse embryonic fibroblasts (MEFs) were isolated and immortalized as described previously (46
). AHPN and 3-Cl-AHPC were emulsified in 25% cremophor EL-75% water and were given by oral or intraperitoneal (i.p.) injection. A dose of 10 to 20 mg/kg body weight was given each day for 1 to 7 days. Protocols for animal use were approved by the Institutional Animal Care and use Committee at the University of Utah.
Chemicals and antibodies.
AHPN and monoclonal antibodies to Flag M2 (catalog number F1804) and β-actin (catalog number A3853) were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). Polyclonal antibodies to poly(ADP-ribose) polymerase (PARP) (catalog number 9532), Bcl-2 (catalog number 2870s), Hsp60 (catalog number 4870s), and hepatocyte nuclear factor 4 alpha (HNF4α) (catalog number 3113s) were obtained from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA). Monoclonal antibodies to cytochrome c (catalog number 556433) and Bcl-2 (catalog number sc-7382) were purchased from BD Pharmingen (BD Biosciences, San Jose, CA) and Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively. Rabbit anti-human SHP was purchased from MBL (catalog number LS-A5411; MBL International Corporation, MA). MitoTracker Red 580 was purchased from Molecular Probes, Inc. (Eugene, OR). Caspase-3 inhibitor (Z-DEVD-FMK; catalog number FMK004), caspase-8 inhibitor (Z-IETD-FMK; catalog number FMK007), and caspase-9 inhibitor (Z-LEHD-FMK; catalog number FMK008) were purchase from R & D (R & D Systems, Minneapolis, MN). Annexin V-phycoerythrin (PE) apoptosis detection kit I was obtained from BD Pharmingen (catalog number 559763; BD Biosciences, San Jose, CA). d-Luciferin was purchased from Xenogen (Xenogen Corporation, Alameda, CA). Flag-SHP plasmid was obtained from Timothy F. Osborne, and hemagglutinin (HA)-HNF4α was obtained from Akiyoshi Fukamizu. LRH-1 and Bcl-2 small interfering RNAs (siRNAs) were purchased from Thermo Scientific Dharmacon RNAi Technologies (NR5A2 ON-TARGET plus SMART pool [L-003430-00-0005] and Bcl-2 ON-TARGET plus SMART pool [L-003307-00-0005]).
Anti-Fas antibody injections and histologic examination.
Two-month-old nontransgenic littermates (NC) or transgenic mice (STG) bred on a C57BL/6 × SJL background were injected i.p. with 10 μg of an affinity-purified hamster monoclonal antibody against mouse Fas antigen (Jo2) diluted in 100 μl of a 0.9-g/liter NaCl solution. Mice were euthanized at specific times after treatment. Tissues were harvested immediately after death, fragments of tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin (H&E).
The terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed using the In Situ Cell Death Detection Kit, Fluorescein (catalog number 11684795910; Roche Applied Science, Mannheim, Germany), following the manufacturer's instructions. In brief, 5-μm cryopreserved tissue sections were fixed in 4% paraformaldehyde for 20 min at room temperature (RT), washed with PBS, and permeabilized with freshly prepared 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. After washing with PBS, the slides were overlaid with 100 μl of TUNEL reaction mixture, according to the manufacturer's instructions, and incubated for 1 h at 37°C. Finally, cells were washed in PBS, and coverslips were mounted onto slides with Prolong Gold antifade reagent with DAPI (4′,6′-diamidino-2-phenylindole) (catalog number p36931; Molecular Probes, Inc., Eugene, OR). Cells were imaged using an Olympus AX70 fluorescence microscope. The magnifications of the photographs of the histology are ×20.
For UV-induced apoptosis, subconfluent fibroblasts growing in the logarithmic phase were subjected to UVC (40 J/m2) irradiation. Cells were fixed, and apoptotic cells were identified by TUNEL assays (Roche). To examine tumor necrosis factor alpha (TNF-α)-induced apoptosis, cells were treated with 10 μg/ml cycloheximide (CHX) plus 30 ng/ml TNF-α at the indicated times and examined for apoptotic cell death by annexin V staining. For AHPN- and 3-Cl-AHPC-induced apoptosis, cells were treated with both agents for 24 h at different doses as indicated. For annexin V staining, cells were washed twice with cold PBS and resuspended in binding buffer provided in the annexin V-PE apoptosis detection kit I at a concentration of 106 cells/ml. A total of 105 cells were then stained with 5 μl annexin V-PE and 5 μl 7-aminoactinomycin D (7-AAD) and incubated for 15 min at RT in the dark. A total of 30,000 cells were acquired by flow cytometry (FACSCalibur; Becton Dickinson) and analyzed with CellQuest software (BD Bioscience).
Immunofluorescence and confocal laser scanning microscopy.
Cells were cultured overnight on coverslips and were treated with various agents as required. Cells were washed twice with PBS, fixed in 3.7% paraformaldehyde (in PBS) for 5 min at RT, and then permeabilized in chilled methanol for 3 min at RT. After washing with PBS, the cells were incubated with blocking solution containing 1% bovine serum albumin (BSA)-PBS for 1 h at RT. To identify SHP protein, cells were incubated first with anti-SHP IgG antibody (3 μg/ml; MBL) diluted in 1% BSA-PBS for 2 h at RT and then with the corresponding Alexa Fluor 488 goat anti-rabbit IgG (1:200; Molecular Probes) as the secondary antibody. To identify Bcl-2, cells were incubated with anti-Bcl-2 IgG antibody (2 μg/ml; Santa Cruz), followed by the corresponding Alexa Fluor 350 goat anti-mouse IgG (1:200; Molecular Probes). To localize mitochondria, cells were incubated with MitoTracker Red 580 at 200 nM in the dark for 20 min at RT. Cells were then washed in PBS, and coverslips were mounted onto slides with Prolong Gold antifade reagent (p36930; Molecular Probes Inc., Eugene, OR). For each sample, confocal images were collected in the fluorescence mode, followed by electronic merging of the images using a confocal microscope (Olympus IX81; Olympus America Inc., Melville, NY).
Cells grown in 100-mm culture dishes were washed three times with ice-cold phosphate buffered-saline (PBS) and collected in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris [pH 8.0], 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with protease inhibitors (catalog number 78410; Thermo Fisher Scientific Inc., Rockford, IL). After a 30-min incubation on ice, cell lysates were sonicated and centrifuged at 14,000 × g for 15 min at 4°C, and the clear supernatants were saved as whole-cell lysates. To isolate the mitochondrion-enriched fraction, cells were washed with PBS, using the mitochondrion isolation kit's protocol (catalog number 89874; Thermo Fisher Scientific Inc., Rockford, IL). For nuclear fractionation, the nuclear and cytoplasmic extraction kit (catalog number 78833; Thermo Fisher Scientific Inc., Rockford, IL) was used. Protein concentrations were determined using the DC protein assay (catalog number 500-0112; Bio-Rad, Richmond, CA). Cell lysates (20 to 40 μg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes according to standard procedures. Membranes were washed in Tris-buffered saline containing 0.05% Tween 20 (TBST), blocked for 1 h with TBST containing 5% nonfat milk, and then incubated with primary antibodies at a 1:1,000 dilution in TBST containing 5% nonfat milk overnight at 4°C. Membranes were then washed with TBST before incubation with horseradish peroxidase-conjugated secondary antibody (3 μg/liter in TBST containing 5% nonfat milk) for 1 h at RT. Membranes were washed four times with TBST before antibody binding was visualized with SuperSignal West Femto maximum-sensitivity substrate (catalog number 34095; Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer's protocol. Equal loading of whole lysate protein, nuclear protein, and mitochondrial protein was verified with β-actin, PARP, and Hsp60, respectively.
For coimmunoprecipitation (co-IP), 1% NP-40 lysis buffer was used instead of RIPA buffer. Protein concentrations of the lysates were quantified by DC protein assay (catalog number 500-0112; Bio-Rad, Richmond, CA). Two hundred micrograms of mitochondrion-enriched lysate was incubated overnight with 1 μg anti-Bcl-2 antibody (Cell Signaling) with agitation at 4°C. The resulting immune complex was precipitated by adding 30 μl Dynabeads M-280 sheep anti-rabbit IgG (SKU number 112-03D; Invitrogen Dynal As, Oslo, Norway) with agitation at 4°C for 2 h, washed four times with the lysis buffer, subjected to 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was probed with the anti-Flag antibody (1:1,000; Sigma).
Colorimetric assay of caspase activities.
Measurements were performed using a kit for each caspase (catalog numbers K106-100, K113-100, and K119-100; BioVision, Inc., Mountain View, CA), in accordance with the manufacturer's instructions. The kits detect specific colorimetric substrates which, when cleaved, increase light absorption at 405 nm. Briefly, cells were cultured overnight on 60-mm culture dishes and then treated with various agents as required. Cells were resuspended in cell lysis buffer provided in the kits and incubated on ice for 10 min, and then cell lysates were centrifuged at 10,000 × g for 1 min at 4°C to collect the supernatants. Protein concentrations were determined using the DC protein assay (catalog number 500-0112; Bio-Rad, Richmond, CA). Two hundred micrograms of protein for each assay was incubated with reaction buffer and substrate at 37°C for 2 h and then measured in a microtiter plate reader.
Athymic mouse pancreatic cancer xenograft studies.
Athymic 5-week-old female nu/nu mice were injected intraperitoneally (i.p.) with 106 BxPc-3 cancer cells for 1 week. Mice were then randomized to oral treatment with either AHPN (10 mg/kg body weight), 3-Cl-AHPC (20 mg/kg body weight), or an equal volume of vehicle (cremophor EL) daily for 5 days. A mixture of 10 μl/mg of firefly d-luciferin (15 mg/ml) (Xenogen), 200 μl ketamine HCl, and 20 μl xylazine (100 mg/ml) was injected i.p. immediately prior to bioluminescent imaging to anesthetize the mice and provide a substrate for the luciferase-expressing cancer cells. The peritoneal tumor implants were imaged with the Xenogen bioluminescent imaging system. The tumor implants for each mouse were verified by microscopy and histopathology.
Mitochondrial respiration assay.
Mitochondrial oxygen consumption and ATP production were measured using techniques described previously (31
). Isolation of mitochondria was performed in livers from 10-week-old male mice. Mice were killed by cervical dislocation. The livers were immediately placed in ice-cold STE buffer (250 mM sucrose, 5 mM Tris, 2 mM EGTA at pH 7.4) and cut into small pieces. After homogenization four times, samples were centrifuged at 1,000 × g
for 3 min and the pellet discarded. The resultant supernatant was then centrifuged three times at 12,000 × g
for 10 min. The isolated mitochondria were resuspended in 500 μL STE buffer, and the concentration of mitochondria was determined using the Bradford protein assay (500-0203; Bio-Rad, Hercules, CA). Rates of oxygen consumption by mitochondria were measured with a FOXY-R-AF probe (Ocean Optics Inc., Dunedin, FL). Measurements were performed with two independent substrates in respiration buffer containing 120 mM KCl, 5 mM KH2
, 1 mM EGTA, 1 mg/ml BSA, and 3 mM HEPES, pH 7.2, at 25°C:. Substrates used were (i) 0.02 mM palmitoyl-carnitine and 5 mM malate or (ii) 5 mM succinate and 0.01 mM rotenone. Oxygen consumption rates were expressed as nmol of O2
·mg dry weight−1
. Respiratory parameters were defined as follows. Basal respiration rates before the addition of ADP (V0
) were defined as state 2. Maximal ADP (1 mmol/liter)-stimulated respiration rates (VADP
) were defined as state 3, and respiration rates in the absence of ADP phosphorylation and measured in the presence of 1 μg/ml oligomycin (Voligomycin
) were termed state 4. Respiration assays were performed in triplicate. The ATP concentration was determined by a bioluminescence assay based on the luciferin/luciferase reaction with the ATP assay kit (ThermoLabsystems).
Detection of ROS.
Production of reactive oxygen species (ROS) was measured using 2′,7′-dichlorofluorescein diacetate (H2DCFDA) (Sigma, D6883). Huh7 cells were grown in 60-mm dishes and loaded with 20 μM H2DCFDA for 2 h at 37°C. After washing twice with ice-cold PBS, cell fluorescence was immediately analyzed using a FACScan flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, CA).
Biochemical analysis of Flag-SHP localization in mitochondria. (i) Subcellular fractionations.
Twenty micrograms of Flag-SHP plasmid DNA was transfected into Huh7 cells in a 15-cm dish. The transfected cells were harvested and disrupted in isotonic buffer (PBS containing 0.2 M mannitol, 0.07 M sucrose, and 1 mM EDTA) containing protease inhibitors (catalog number 78410; Thermo Fisher Scientific Inc., Rockford, IL), followed by centrifugation at 800 × g at 4°C for 10 min to obtain postnuclear supernatant (PNS). PNS was centrifuged at 17,000 × g at 4°C for 10 min to obtain the mitochondrion-enriched precipitate fraction (P1). The supernatant was centrifuged at 170,000 × g at 4°C for 30 min to separate the microsome-enriched precipitate (P2) and supernatant (S) fractions. The subcellular fractions were separated by SDS-PAGE and then analyzed by Western blotting.
(ii) Alkaline treatment of mitochondria.
One hundred micrograms of mitochondrial protein was prepared from the Huh7 cells expressing Flag-SHP protein and treated with 100 mM Na2CO3 in 10 times the volume of mitochondrial suspension for 1 h on ice. The reaction mixtures were centrifuged at 170,000 × g at 4°C for 30 min to separate the precipitate and supernatant fractions. The fractions were subjected to SDS-PAGE followed by Western blot analysis.
(iii) Trypsin protection assay.
The mitochondrion were treated with either H2O or 2% Triton X-100 in 10 times the volume of mitochondrial suspension on ice for 1 h and then treated with 50 μg/ml trypsin on ice for 1 h. The reaction mixtures were separated by SDS-PAGE and then analyzed by Western blotting.
All the experiments were repeated at least three times, and error bars represent the standard errors of the means (SEM). Statistical analyses were carried out using Student's unpaired t test; a P value of <0.01 was considered statistically significant.