 wash cells twice with cold PBS and then resuspend cells in 1 × Binding Buffer at a concentration of 1 × 10 6 cells/mL. Add 5 µL of FITC Annexin V and 5 µL PI, then Gently vortex the cells and incubate for 15 min at RT in the dark. Stained cells were analyzed by flow cytometry and quantified using the CellQuest software. At least 10,000 events were analyzed and compared with control. For DAPI staining, treated cells were fixed in pre-chilled 3.7% methanal for 10 min and then stained with 0.1 μg/mL of 4-6-diamidino-2-phenylindole (DAPI) for 10 min. Nuclei were examined and imaged using a fluorescence microscope after being washed three times.
RNA extraction and quantitative real-time PCR (qPCR). Total RNA was extracted with a TRIzol reagent (Invitrogen, USA). cDNA was synthesised using the HiFi-MMLV cDNA Kit (Cwbiotech, China). PCR analyses were performed with Forget-Me-Not ™ EvaGreen ® qPCR Master Mix (Biotium, USA). The primers used are shown in Supplementary Table S1. The data were normalised to actin expression and further normalised to the negative control. The fold changes were calculated through relative quantification (2 −ΔΔCt ).
Wound-healing assay. The migratory ability of the SMMC-7721 and HepG2 cells was measured with a wound-healing assay. Cells were seeded at a density of 5 × 10 5 cells per well on a 12-well plate and grown to about 90% confluence after 24 h. The medium was then removed, and the cells were wounded by manually scraping them with a plastic pipette tip. The cells were washed twice with PBS, and then they were incubated with fresh medium in the presence of JNU-144 or DMSO. To monitor the cell migration, images were captured immediately at 0, 24 and 48 h post wounding with a microscope.
In vitro migration and invasion assay. Cells were treated with DMSO or JNU-144 for 12 h. Subsequently, they were collected and resuspended in serum-free media at a density of 1 × 10 6 /mL and plated at 100 μL cell suspension in chambers with 8 μm pores (Corning, USA). These chambers were put in wells containing fresh media supplemented with 10% FBS. After incubation for 24 h at 37 °C in 5% CO 2, the cells on the upper surface of the filter were wiped with a cotton swab. Cells that invaded the lower surface of the filter were fixed by 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. Cell numbers were counted in six random optical fields (×10) per filter under a microscope. A cell invasion assay was conducted in a similar model, except the transwell membrane was pre-coated with 100 μg Matrigel (Corning, USA). Immunofluorescent staining. Cells cultured on coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 10 min after treatment as indicated. The cells were then incubated with PBS containing 0.25% Triton X-100 for membrane permeabilisation and then blocked with 5% bovine serum albumin (BSA) for 1 h. Next, the cells were incubated with specific primary antibodies overnight at 4 °C. Then, the coverslips were washed three times with PBS, followed by incubation for 1 h in the dark with fluorescent secondary antibodies (Invitrogen, USA). Finally, the coverslips were stained with 4-6-diamidino-2-phenylindole (DAPI) for 10 min and captured by a fluorescence microscope.
Mouse xenograft models. Four-to six-week-old female BALB/c nude mice were obtained from Guangdong Medical Lab Animal Center. To establish the liver tumor xenograft mouse models, 5 × 10 6 SMMC-7721 cells in 200 μL saline were implanted subcutaneously into the right flank of the nude mice. After implantation, tumor volumes were measured with a slide calliper every 2 days and calculated using the following formula: 0.5 × (length) × (width) 2. When the tumor size reached 100 mm 3, the tumor-bearing mice were divided randomly into two groups and treated with the vehicle (1% DMSO in saline) or 10 mg/ kg JNU-144 in saline every 2 days by i.p. injection. The mice were sacrificed after six times injection, and tumor tissues were excised, weighed and photographed. Subsequently, xenograft tumor and the major organs were fixed in neutral buffered formalin for western blot analysis, immunohistochemistry and pathological examination.
All of the animals were treated according to protocols approved by Institutional Animal Care and Use Committee of the Shenzhen People's Hospital. And this study was approved by Institutional Animal Care and Use Committee of the Shenzhen People's Hospital (Approval Document No. LL-KT-201701017).
Haematoxylin eosin (H&E) staining and immunohistochemistry. Briefly, fixed tissues and organs were embedded in paraffin. After being cut into 5 mm slices, the sections were stained using a standard H&E procedure. The results were analysed under a phase-contrast Olympus microscope (Olympus America, Inc.). For the immunohistochemical analysis, deparaffinised tumor sections underwent antigen retrieval in sodium citrate buffer (pH 6.0) using the high-pressure method. Then, the slides were blocked with 5% BSA and incubated with specific primary antibodies, followed by incubation with streptavidin-peroxidase horseradish peroxidase conjugated secondary antibodies and stained with a Dako kit (Dako, USA). The sections were counterstained with haematoxylin and analyzed under a phase-contrast Olympus microscope. Statistical analysis. Data were analyzed using GraphPad Prism 6.0 software (GraphPad software Inc., USA).
The differences between the two groups were estimated with a two-tailed unpaired t-test. The quantitative data are reported as means ± SD from at least three independent experiments. P values less than 0.05 were considered statistically significant.

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<h2>Summary function for pyears objecs</h2>

<h3>Description</h3>

<p>Create a printable table of a person-years result.</p>


<h3>Usage</h3>

<pre>
## S3 method for class 'pyears'
summary(object, header = TRUE, call = header, n = TRUE,
event = TRUE, pyears = TRUE, expected = TRUE, rate = FALSE, rr =expected,
ci.r = FALSE, ci.rr = FALSE, totals=FALSE, legend = TRUE, vline = FALSE,
vertical= TRUE, nastring=".", conf.level = 0.95,
scale = 1,...)
</pre>


<h3>Arguments</h3>

<table summary="R argblock">
<tr valign="top"><td><code>object</code></td>
<td>
<p>a pyears object</p>
</td></tr>
<tr valign="top"><td><code>header</code></td>
<td>
<p>print out a header giving the total number of
observations, events, person-years, and total time (if any)
omitted from the table</p>
</td></tr>
<tr valign="top"><td><code>call</code></td>
<td>
<p>print out a copy of the call</p>
</td></tr>
<tr valign="top"><td><code>n, event, pyears, expected</code></td>
<td>
<p>logical arguments: should these
elements be printed in the table?</p>
</td></tr>
<tr valign="top"><td><code>rate, ci.r</code></td>
<td>
<p>logical arguments: should the incidence rate and/or
its confidence interval be given in the table?</p>
</td></tr>
<tr valign="top"><td><code>rr, ci.rr</code></td>
<td>
<p>logical arguments: should the hazard ratio and/or
its confidence interval be given in the table?</p>
</td></tr>
<tr valign="top"><td><code>totals</code></td>
<td>
<p>should row and column totals be added?</p>
