Supplemental Information
Figure legends
Fig S1. UK-101 induces G1 cell cycle arrest in PC-3 cells. A. The cell cycle distribution was analyzed 24
hours after UK-101 treatment using flow cytometry.
An increase in the proportion of cells in the G1
phase of the cell cycle was seen, suggesting G1 cell cycle arrest. [%CV (peak widths) was between 5.44
and 6.41 for all conditions]
B. Apoptosis was analyzed 24 hrs after UK-101 treatment by staining with
PI and AV (as in Fig 4B). A minimal increase in late apoptosis was seen, with no significant increase in
early apoptosis. Numbers are means plus or minus standard deviations. C. To verify accumulation of
cells in the G1 phase of the cell cycle, p27 accumulation was examined via western blot. Both UK-101
and epoxomicin increased the amount of p27 in cells compared with vehicle.
Fig S2. UK-101 treatment does not result in significant systemic toxicity in PC-3 tumor bearing nude
mice.
A. In contrast to mice treated with cisplatin, UK-101-treated mice appeared to suffer significantly
less systemic toxicity (based on body weight and death rate). Pictures were taken every week over three
three weeks. B. The weights of mice treated with UK-101 also remained steady over the 21 day treatment
period.
Materials and Methods
Immunoblotting. Whole cell lysates were prepared by incubating cells in non-denaturing lysis
buffer (50nM Tris-Cl, 150mM NaCl, 1% NP-40, 1% Triton X-100, and 1% protease inhibitor
cocktail (Sigma-Aldrich, St. Louis, MO)) on ice for 1 hour. Cells were then centrifuged at
14,000 rpm for 10 min at 4°C (Sorvall Biofuge Primo R, Kendro Laboratory Products, Newtown,
CT). Supernatants were collected and subjected to protein assay via method of Bradford using
Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA) and a GENESYS 10
spectrophotometer, Thermo Spectronic (VWR, Arlington Heights, IL). The supernatants were
then mixed with 2x Laemmli Sample Buffer (Sigma-Aldrich) and heated in boiling water for 10
min. Subsequently, the denatured whole cell lysates were resolved by 8%-12% SDS-PAGE and
transferred to PVDF membranes (Bio-Rad). Membranes were blocked with 5% dry skim milk
(Bio-Rad) or BSA (Sigma-Aldrich) for 1 hour at room temperature on the rotator. Appropriate
primary and secondary antibodies were used to incubate the membranes for 1 hour at room
temperature on the rotator or overnight at 4oC. Finally, Amersham ECL Western Blotting
Detection Reagents (GE Healthcare Life Sciences, Pittsburgh, PA) were used to visualize
proteins of interest on Kodak BioMax XAR Films (Sigma-Aldrich).
Nuclear and cytoplasmic protein extraction (Adapted from a protocol kindly provided by Dr. E.
Penni Black (University of Kentucky)). Cells were trypsinized and pelleted for 5 minutes at 5000
rpm in a 4oC centrifuge. The supernatant was aspirated and cells were stored at -80oC until lysis.
After thawing, cells were quick spun (10 sec, 6000 rpm) to allow the removal of any residual
media. The cytoplasmic lysis buffer (10mM HEPES pH 7.9, 10mM KCl, 3mM MgCl2, 0.05%
NP-40, 1mM EDTA pH 8.0, 10mM beta-glycerolphosphate, 0.5mM NaF, 0.5mM NaVO4, 1mM
2
DTT, and 1% protease inhibitor cocktail) was added at a volume ten times that of the pellet, and
cells were allowed to swell on ice for 30 minutes. The solutions were spun down (5 minutes at
5000 rpm in a 4oC centrifuge) and 80% of the supernatant volume was collected as the
cytoplasmic lysate. Pellets were resuspended in the cytoplasmic lysis buffer, spun down as
before, and all the supernatant was removed and discarded. Pellets were then resuspended in the
nuclear lysis buffer (50mM HEPES pH 7.9, 250mM KCl, 0.1% NP-40, 0.1mM EGTA pH 8.0,
0.1mM EDTA pH 8.0, 10% glycerol, 10mM beta-glycerolphosphate, 0.5mM NaF, 0.5mM
NaVO4, 1mM DTT, and 1% protease inhibitor cocktail) at half the volume as the cytoplasmic
lysis buffer and allowed to lyse for 30 minutes on ice. The solutions were then spun down for 10
minutes at 14000 rpm in a 4oC centrifuge, and the supernatant was collected as the nuclear
fraction. Protein concentrations were determined by the method of Bradford, and samples were
denatured in 2X Laemali sample buffer, as above.
Immunofluorescence. Coverslips were sterilized with ethanol and UV light in a 35mm dish.
For the interferon-gamma conditions, cells were plated onto the dishes and treated as indicated.
At the end of the treatments, cells were fixed with 4% paraformaldehyde in PBS and
permeabilized with 0.2% Triton-X in PBS (PBST). Coverslips were then blocked with 10%
serum and 1% BSA in PBST. Primary antibody was added in DackoCytomation Antibody
Diluent with Background Reducing Components at 1:800. Subsequently, secondary antibodies
were added (AF488 at 1:1000 and Rhoadmine Phalloidin at 1:1000) in the same antibody diluent.
Coverslips were washed well in PBST, mounted onto slides with Prolong Gold antifade reagent
with DAPI, and allowed to solidify overnight. The next day, the coverslips were sealed and
visualized.
siRNA. Briefly, PC-3 cells were plated at a density of 20,000 cells per well in a 24 well plate and
allowed 24 hours to attach. The siRNA was diluted separately from the DharmaFECT2® and,
after a five-minute incubation at room temperature, the two were combined. They were then
allowed to incubate for an additional 20 minutes prior to the addition of complete media. The
final solution added to the cells contained 100nM siRNA and 0.5µL of DharmaFECT2® per
well. 24 hours post-transfection, the transfection media was removed and fresh media was added.
Media was refreshed every 24 hours and cells were subcultured when the untreated and mock
transfected controls reached >70% confluence. Samples were collected at the time points
indicated and subjected to western blotting analysis as detailed above.
Cell counting. After trypsinization, each well of cells was resuspended in an equal volume of
media. An aliquot was taken and mixed one to one with trypan blue. 10µL of this cell solution
was then added to a TC10 dual-chamber counting slides, in duplicate, and counted on a TC10™
Automated Cell Counter (Bio-Rad). The number of viable cells per milliliter was used to
determine the total viable cell population from each well, and this was plotted using GraphPad
PRISM® (GraphPad Software, Inc, La Jolla, CA).
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Cell viability assay. The indicated inhibitors were added in increasing concentrations, and cells
were treated for 48 hours. Briefly, 20µL of MTS reagent was added to cell samples in 100µl of
culture media, and cells were incubated for 1 hour at 37°C. Absorbance was recorded at a
490nm wavelength on a microplate reader (FL600; Bio-Tek Instruments, Inc., Winnoski, VT)
using the software KC4 v.2.5 (Bio-Tek Instruments, Inc., Winooski, VT). Cell proliferation was
determined as a percentage relative to DMSO (vehicle) treated cells. IC50 values were calculated
from sigmoidal dose response curves obtained by nonlinear regression using GraphPad PRISM.
These data represent the average of three or more replicates.
Chymotrypsin-like (CT-L) cell-based activity assay. PC-3 cells were plated on a white-walled
96-well plate at a density of 8,000 cells/well in 50μL media/well. Cells were incubated at 37°C
and 5% CO2 for 24 hours prior to treatment with either vehicle control (DMSO), inhibitor
control (10μM epoxomicin), or increasing concentrations (1μM, 10μM, and 50μM UK-101;
5nM, 50nM, 500nM epoxomicin or bortezomib) of proteasome inhibitors for 2hrs. Following
equilibration to room temperature, 50μL of Proteasome-Glo Cell-Based Reagent (Promega)
containing the luminogenic proteasome substrate Suc-LLVY-aminoluciferin was added to each
well. Samples were mixed at 400rpm for 4min on a plate shaker and incubated at room
temperature for 10min. Luminescence was then recorded for each sample using a Veritas™
Microplate Luminometer (Promega Corp., Sunnyvale CA).
Flow Cytometry. Cells were treated with drug for 24 or 48 hours and both floating and adherent
cells were collected. Cells were pelleted by centrifugation at 600g-1 for 10 minutes at 4°C. After
two washes in cold phosphate buffered saline (PBS), the pellet was resuspended in 1X Binding
Buffer at 1x106 cells/mL and a 500 µL aliquot of the cell suspension was transferred to a test
tube with 5µL of Annexin V-FITC and 10µL propidium iodide (PI). This solution was allowed
to incubate, protected from light, for 10 minutes at room temperature prior to analysis by flow
cytometry (Becton-Dickinson FACScalibur). At least 2x104 cells were analyzed per sample,
categorized as viable (Annexin-V and PI negative), apoptotic (Annexin-V positive and PI
negative).
Flow cytometry was performed for cell cycle analysis using assay kits (Invitrogen) following
instructions provided by manufacturers. Cells were collected as above with centrifugation for six
minutes instead of ten. They were washed with cold PBS and then resuspended in 0.5mL cold
PBS. 4.5mL of 70% ethanol on ice was used as a fixative, and the PBS cell solution was mixed
with the fixative and incubated for at least two hours prior to centrifugation. The cells were
spun at 200g-1 for 5 minutes at 4°C. After carefully decanting the supernatant, the pellet was
resuspended in cold PBS and centrifuged again a minute later. The final cell pellets were
resuspended in 1mL PI/Triton X-100 staining solution with RNAse A (2mg DNAse-free RNAse
A (Sigma-Aldrich) and 200µL of 1mg/mL PI in 100mL of 0.1% (v/v) Triton X-100 in PBS) and
incubated for 30 minutes at room temperature prior to analysis by flow cytometry. A minimum
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of 1 x 104 cells were analyzed per sample. The DNA histogram was further analyzed using
integration software (ModFit LT V2.0, Topsham, MN, USA) for cell cycle analysis.
Caspase activity assay. Briefly, cells were plated on sterile white-walled 96-well plates and
treated in the same manner as for the MTS assay. After equilibrating to room temperature,
100µL of the assay dye was added to each well and mixed thoroughly (400 rpm for 30 sec
[Caspase 3/7] or 2 min [Caspase 8 & 9]). After an additional 30 minute incubation,
luminescence was measured for each sample using a Veritas™ Microplate Luminometer
(Promega Corp., Sunnyvale CA). Luminescence was normalized to control, plotted, and tested
using one-way ANOVA in GraphPad PRISM® (GraphPad Software, Inc, La Jolla, CA). These
data represent the average of three or more replicates.
Antitumor activity study in in vivo xenograft animal model. Six-week-old male BALB/c athymic
nude mice were purchased from Japan SLC (Hamamatsu, Japan). All mice were housed in a
room that was automatically maintained at 21–25°C and relative humidity (45–65%) with a
controlled light–dark cycle. PC-3 human prostate cancer cells were injected subcutaneously (1 ×
107 tumor cells/0.1 ml of serum-free RPMI 1640 medium/animal) with a 27-gauge needle into
the right lower flanks in carrier mice. After 20 days, when the tumors had reached an average
volume of 300-400 mm3, the tumor-bearing nude mice were intraperitoneally (i.p.) injected with
UK-101 (1 mg/kg and 3 mg/kg dissolved in 0.05% DMSO in saline) twice per week for 3 weeks.
Cisplatin (2 mg/kg, i.p.) (cisplatin injection 10 mg/20 ml, Choongwae Pharmaceutical Corp.,
Seoul, Korea) was also injected twice a week as a positive control. The group treated with
0.05% DMSO in saline was designed as the control. The weights and tumor volumes of the
animals were monitored twice per week. The tumor volumes were measured with vernier
calipers (Mitutoyo Co., Kawasaki, Japan) and calculated by the following formula: (A × B2)/2,
where A is the larger and B is the smaller of the two dimensions. At the end of the experiment,
the animals were sacrificed by cervical dislocation. The tumors were separated from the
surrounding muscles and dermis, excised and weighed.
For immunohistochemical analysis, all specimens were fixed in formalin and paraffin-embedded
for examination. Sections of 5 μm thickness were cut on a microtome (Leica Co., Nussloch,
Germany). Paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled
water, and then subjected to heat-mediated antigen retrieval treatment using a 10 mM sodium
citrate buffer. Endogenous peroxidase activity was quenched by incubation in 2% hydrogen
peroxide in methanol for 15 minutes and then clearing with PBS three times for 5 minutes. The
sections were blocked for 30 min with 3% normal horse serum diluted in PBS; the sections were
then blotted and incubated with rabbit cleaved-caspase-3 polyclonal antibody (1:200 dilution,
Cell Signaling Inc., Beverly, MA) in blocking serum overnight at 4°C. The next day, slides were
washed three times for 5 minutes each in PBS and incubated in biotinylated anti-rabbit antibody
for 1 h. The slides were washed in PBS, followed by addition of the avidin-biotin-peroxidase
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complex (ABC, Vector Laboratories, Inc., Burlingame, CA). The slides were washed again, and
the peroxidase reaction was developed with diaminobenzidine and peroxide. The slides were
then counterstained with hematoxylin, mounted in aqua-mount, and evaluated by light
microscopy (200X, Olympus, Tokyo, Japan). A negative control was performed in all cases by
omitting the primary antibody.
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