Supplementary methods
Immunocytochemistry
The immunocytochemical analysis was performed with cultured HLF cell on
glass slides coated with saline in the presence of concentration of
AZD1152-HQPA (100nM) for 72 h. The cell was fixed using 100% methanol for
20 min at -20 °C, and then blocked in PBS-3 % BSA for the immunocytochemical
detection. The primary antibody for Bcl-xL (Cell Signaling technology, Beverly,
MA, USA, Catalog No.#2764) or Bcl-2 (Santa Cruz Biotechnology, Dallas, TX,
USA, Catalog No.#sc-783) were used at 1:200 dilution. Control γ-tubulin
antibody (Sigma, Catalog No.T6557) was used at 1:1000. The secondary
antibody for Bcl-xL or Bcl-2 was the Alexa Fluor® 568 fragment of a donkey
anti-rabbit IgG (H+L), and the secondary antibody for γ-tubulin was the Alexa
Fluor® 488 fragment of a donkey anti-mouse IgG (H+L) (Invitrogen). Cellular
DNA was counterstained with 4’,6-dia-midino-2-phenylindeole. After mounting,
the slides were visualized with a fluorescent microscope (Axio Observer.ZL, Carl
Zeiss, oberkochen, Germany).
Time-lapse analysis
HLF cell was plated separately at a density of 5x104 cells in 6-cm dishes (Sigma)
and in log-growth phase in DMEM medium (Invitrogen), supplemented with 5%
FBS and Pen/Strep (Sigma) as antibiotics. After incubation in 5% CO 2 at 37°C
overnight, HLF cell treated with AZD1152-HQPA 100nM for 72h. After washout
by replacing the medium with drug-free growth medium (AZD1152 washout) or
further treated with 1μM ABT263, HLF cell medium attachment was confirmed
for 24h. Image analysis was performed using Axio-Vision and Axio-Observer
(Carl Zeiss).
Apoptosis assay
Cells were plated at 0.8x105/ml in 10cm dishes and incubated with
AZD1152-HQPA (100nM), ABT263 (1μM), or AZD1152-HQPA (100nM) followed
by ABT263 (1μM). For detection of apoptosis, cells were labeled with Annexin V
staining (the Annexin V-FITC Kit (Miltenyi Biotec, Gladbach, Germany; Catalog
No.130-092-052) and 7-ADD (BD Pharmingen, San Diego, CA, USA; Catalog
No.51-68981E). The Annexin V assays were performed according to the
manufacture’s protocol (BD Pharmingen). Briefly, the cultured cells were
collected, washed with binding buffer, and incubated in 100μl of a binding buffer
containing 5μl of Annexin-V-FITC. The nuclei were counterstained with
7-ADD.The percentage of apoptotic cells was determined using FACS Canto™ II
flow cytometer, followed by analysis on a FACS Canto™ (BD Biosciences, San
Jose, CA, USA) using FACS Diva software(BD Biosciences). Expression of
cleaved capase-3 was analyzed by Western blotting using anti-cleaved
caspase-3 (1:200; Cell Signaling; Catalog No.9661).
In vitro assay of cell proliferation and viability
All cell lines of human HCC were cultured in logarithmic growth phase in the
presence of various concentrations of AZD1152-HQPA or ABT263. Cells were
seeded at 5x104 cells in twelve-well plates (Sigma) with the appropriate control
medium. After 24h, plates were treated with compound and incubated at 37°C.
At the end of the incubation time, cells were detached from each plate, and
viable cells were counted using a hemocytometer. Half-maximal inhibitory
concentration (IC50) values were calculated with BioDataFit v.1.02 software
(Chang Bioscience, Castro Valley, CA, USA) using the four-parameter logistic
model. The number of viable cells was counted by an automatic cell counting
machine (CYTORECON; GE Healthcare, Cardiff, UK) according to the
manufacturer’s instructions. The mean values and standard deviations of IC50
were calculated in triplicate for each cell line.
To investigate viability, cells were
cultured with AZD1152-HQPA (0.001-1000nM) for 72 h, and/or ABT263
(0.001-100μM) for 24h. The number of nonviable cells was assessed using
CYTORECON and trypan blue (Sigma) dye exclusion. These experiments were
independently evaluated in triplicate for cell proliferation analysis.