Supplementary Materials and Methods
Cell lines, primary AML cells, reagents and antibodies
U937, HL-60, and Jurkat cells were provided by American Type Culture Collection
(ATCC, Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10%
fetal bovine serum (FBS).
Peripheral-blood samples for the in vitro studies were obtained from 6 patients
with newly diagnosed or recurrent acute myeloid leukemia (AML) after informed
consent. Approval was obtained from the Southwest Hospital (Chongqing, China)
institutional review board for these studies. AML blasts were isolated by density
gradient centrifugation over Histopaque-1077 (Sigma-Aldrich Co., St Louis, MO) at
600g for 15 minutes. Isolated mononuclear cells were washed and assayed for total
number and viability using trypan blue exclusion. Cells were suspended at 8105/mL
in RPMI 1640 medium for treatment.
Ursolic acid (UA) was purchased from Sigma (St Louis, MO). Y-27632 was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Z-VAD-FMK was from
EMD Biosciences (La Jolla, CA). Antibodies against Ezrin, phospho-EzrinTyr353,
Moesin, Cleaved-Caspase-3, and Caspase-8 were purchased from Cell Signaling
(Danvers, MA); Rho, ROCK1, GAPDH and FasAPO-1-1 were from Santa Cruz
Biotechnology (Santa Cruz, CA); FADD was from PharMingen (San Diego, CA); and
PARP was from Biomol (Plymouth Meeting, PA).
LC-ESI-Q-TOF MS/MS analysis and protein identification
The total cellular samples were lysed in a buffer containing: 8 M urea, 2 M thiourea,
2% 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), 65 mM
DTT, 1% nuclease mix, 1 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 10 μg/ml
leupeptin, 1 mM PMSF. Equal amounts of protein (70 g/sample) were separated by
SDS-PAGE, the gels were stained with Coomassie brilliant blue G-250 and the bands
containing proteins were cut into 40 slices. Each slice was digested with trypsin. The
supernatant peptide were sequentially extracted with peptide extraction solution (5%
formic acid (FA), 67% ACN) and subjected to analysis by LC-ESI-Q-TOF MS/MS
(Agilent, Santa Clara, CA, USA). The analysis of MS/MS data was performed using
Spectrum Mill MS Proteomics Workbench (RevA.03.03.078) against the UniProtKB/
SWISS-PORT Homo sapiens (Human) or Mammals (Bos Taurus) database.
Lentiviral-mediated ezrin-overexpression cells
Human ezrin lentiviral construct was generated by inserting human full-length ezrin
cDNA into Ubi-MCS-3FLAG-IRES-puromycin lentiviral vector (GeneChem,
Shanghai, China). The human ezrin lentiviral expression plasmid or GFPpuromycin-LV vector was co-transfected into 293Ta cells with the Lenti-Pac HIV
Packaging Mix (GeneChem, Shanghai, China). Lentivirus-containing supernatant
were harvested 48 h after transfection. To establish stable ezrin-overexpressing cell
lines, U937 cells were transduced with serial dilutions of lentiviral supernatant in the
presence of 5 μg/ml polybrene and selected by 5 g/ml puromycin. After antibiotic
selection for 3 weeks, stable overexpressing ezrin cells were obtained.
RNA interference
Oligonucleotides with the following targeting sequences were used for the cloning of
small hairpin RNA (shRNA)-encoding sequences in hU6-MCS-Ubiquitin-EGFPIRES-puromycin lentiviral RNAi vector: ezrin, 5’-CCTGGAAATGTATGGAATC
AA-3’. After co-transfection of lentiviral packaging plasmids into 293Ta cells,
lentivirus-containing supernatant were harvested 48 h after transfection. U937 cells
were transduced with serial dilutions of lentiviral supernatant in the presence of 5
μg/ml polybrene and selected by 5 g/ml puromycin. After antibiotic selection for 3
weeks, stable ezrin-siRNA cells were obtained.
Immunoblotting
The total cellular samples were lysed in 1 NuPAGE LDS sample buffer
supplemented with 50 mM dithiothreitol. The protein concentration was determined
using Coomassie Protein Assay Reagent (Pierce, Rockford, IL). For SDS-PAGE,
proteins were loaded at 30 μg and transferred to nitrocellulose membrane. Membranes
were blocked with 5% fat-free dry milk in 1 Tris-buffered saline (TBS) and
incubated with antibodies. Protein bands were detected by incubating with
horseradish peroxidase-conjugated antibodies (Kirkegaard and Perry Laboratories,
Gaithersburg, MD) and visualized with enhanced chemiluminescence reagent (Perkin
Elmer, Boston, MA).
Apoptosis detection assay
Apoptotic cells were evaluated by flow cytometric analysis using FITC conjugated
Annexin V/propidium iodide (PharMingen, San Diego, CA) staining according to the
manufacturer’s manual. Both early apoptotic (Annexin V-positive, PI-negative) and
late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death
determinations.
Death-inducing signaling complex (DISC) immunoprecipitation
Cells were lysed in 1% NP-40 buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1%
Nonidet P-40, 10% glycerol, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1
mM Na3VO4). Equal quantities of proteins were precleared with non-specific normal
mouse IgG (Pierce Biotechnology, Rockford, IL) and incubated overnight at 4°C with
anti-FasAPO-1-1. Immune complexes were collected with protein G agarose beads
(Pierce Biotechnology, Rockford, IL). Beads were washed five times in lysis buffer,
boiled in sample buffer containing DTT, and proteins were then separated by
SDS-PAGE. Blots were incubated with antibodies against using Ezrin, FADD,
Caspase-8, as well as Fas.
Immunofluorescence
After treatment, cells were collected by centrifugation, attached to glass coverslips
pre-coated with polylysine and fixed with cold-acetone for 30 min, permeabilized
with 0.1% Triton X-100 and blocked for 30 min with 1% BSA in PBS. For
localization of Fas and Ezrin, cells were incubated with anti-FasAPO-1-1 (Santa Cruz
Biotechnology, CA) and anti-Ezrin (Cell Signaling, Danvers, MA) primary antibodies
at 4°C overnight, followed by the secondary Alexa 488-conjugated goat anti-mouse
antibody and Alexa 647-conjugated donkey anti-rabbit antibody (Molecular Probes,
Eugene, OR) for 1 h at room temperature. After washing, the nuclei were
counterstained with 0.1 μg/ml DAPI (Sigma-Aldrich Co., St Louis, MO) for 5 min.
Samples were then observed using a laser confocal scanning microscope
(LCS-SP2-MP-AOB).
Rho activity assay
Rho activity assays were performed according to the manufacturer’s instructions
(Calbiochem, Merck KGaA, Darmstadt, Germany). Briefly, 5×105 U937 cells were
plated for 2 days. Samples were rapidly lysed at 4°C and incubated with
Sepharose-bound Rhotekin to specifically pull down active Rho. After washing, the
bead/protein complexes were boiled in sample buffer and then separated by
SDS-PAGE. Blot was incubated with antibody against Rho.
Xenograft assay
NOD/SCID mice (5 weeks old) were purchased from Vital River Laboratories (VRL,
Beijing, China). All animal studies were conducted according to protocols approved
by the Institutional Animal Care and Use Committee (IACUC) of the University.
U937 cells (2106/0.2 mL/mouse) were suspended in sterile PBS and subcutaneously
injected into the right flank of the mice. Mice were randomized into two groups
(n=10). Five days after tumor inoculation, Mice were received UA (50 mg/kg, i.p. for
15 days) or an equal volume of vehicle. Tumor size and body weight were measured
after treatment at various time intervals throughout the study. At the termination of
the experiment, mice were sacrificed at 24 h after the last administration of compound.
The tumors were excised and weighed. Tumors were collected at selected times and
fixed in paraformaldehyde. Paraffin-embedded tissues were sectioned and processed
for H&E, TUNEL and immunohistochemical staining.
TUNEL assay
The apoptotic cells in tissue samples were detected using an In Situ Cell Death
Detection kit (Roche, Mannheim, Germany) according to the manufacturer’s manual.
After deparaffinization and permeabilization, the tissue sections were incubated in
proteinase K for 15 min at room temperature. The sections were then incubated with
the TUNEL reaction mixture that contains terminal deoxynucleotidyl transferase (TdT)
and fluorescein-dUTP at 37C for 1 h. After washing three times with PBS, the
sections were incubated with the Converter-POD which contains anti-fluorescein
antibody conjugated with horse-radish peroxidase (POD) at room temperature for 30
minutes. After washing three times with PBS, the sections were incubated with 0.05%
DAB and analyzed under light microscope.
Histological and Immunohistochemical evaluation
At the termination of experiments, tumor tissues from representative mice were
sectioned, embedded in paraffin, and stained with hematoxylin and eosin for
histopathologic evaluation. For immunohistochemical analysis, tissue sections 4 μm
in thickness were dewaxed and rehydrated in xylene and graded alcohols. Endogenous
peroxidase activity was quenched by incubation in TBS-T containing 3% hydrogen
peroxide. Antigen retrieval was performed with 0.01 M citrate buffer at pH 6.0 for 20
min in a 95C water bath. After blocking with 10% goat serum for 1 h, sections were
incubated with primary antibodies against Cleaved-Caspase-3 and Ezrin, washed three
times in PBS, incubated with biotinylated secondary antibody for 1 h, followed by
incubation with a streptavidin-peroxidase complex for another 1 h. After three
additional washes in PBS, diaminobenzidine working solution was applied. Finally,
the slides were counterstained in DAPI.
Statistical Analysis
Tumor volumes, body weights, and percentage of apoptotic cells were represented as
mean  SD. Statistical analyses were performed using the 2-tailed Student t test. p <
0.05 (* ) or p < 0.01 (**) were considered significant.