Supplementary Materials and Methods
Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Inc. (Kumamoto, JAPAN).
Annexin
V-FITC and PI apoptosis detection kit was obtained from Jiamay Biotech. Bay 11-7082 was
purchased from Sigma-Aldrich. Mouse RelA/p65 (NM_009045) was constructed into pCMV6
vector.
Cell Proliferation
EOMA cells were seeded in flat-bottom 96-well plate at a final density of 6×103 cells per well and
cultured in the growth medium supplemented with 10% FBS. Cells were allowed to adhere and
recover overnight, and then replaced with fresh culture medium (DMEM, 1% FBS). EOM cells were
treated with indicated doses of endostatin. After 48 h, each well were added with 10μl
CCK-8solution and incubated for 2 h. The cell viability was measured in a 96-well format plated
reader (Varioskan Flash Spectral Scanning Multimode Reader, Thermo Scientific) at wavelength of
450 nm.
Flow Cytometry
EOMA cells were seeded in 6-well culture plate at a final density of 9×105 per well. EOM cells were
treated with indicated doses of endostatin, after 48 h, harvested and washed in cold PBS. Annexin V
and PI staining was carried out using the Annexin V-FITC and PI apoptosis detection kit according to
the manufacturer’s protocol. Cells were analyzed by a FACS Calibur flow cytometry system (Becton
Dickinson, San Jose).
Routine Blood Test
Nude mice were inoculated subcutaneously with 1×106 EOMA cells. After seven days, mice
were grouped randomly and treated with 12 mg/kg endostatin or saline intratumorally every other
day for 18 days. Blood samples were collected from orbital sinus and tested by the routine blood test
equipment (Sysmex).
Supplementary Figure legends
Supplementary Figure S1. Endostatin inhibits the tumorigenesis of hemangioendothelioma
Nude mice were inoculated subcutaneously with 1×106 EOMA cells. After seven days, mice were
grouped randomly and treated with 12 mg/kg endostatin or with placebo (saline) intratumorally
every other day for 18 days. The following data were collected: tumor volume (A), tumor weight (B),
Kaplan-Meier survival (C) (n=5), hemoglobin concentration (D) and platelet counts (E) in peripheral
blood (n=7). H&E staining of EOMA sections is shown in (F). The dashed lines and black arrows
indicate hemorrhage. Simultaneously, 1×106 EOMA cells were inoculated subcutaneously into nude
mice along with 12 mg/kg endostatin or placebo (saline). Tumor volume (A) and tumor weight (B)
were evaluated. Data are shown as mean ± SD. All of the aforementioned experiments were repeated
at least twice. (*, P<0.05; **, P<0.01; ***, P<0.005).
Supplementary Figure S2. Endostatin has no effect on EOMA cell proliferation or apoptosis in
vitro
(A) EOMA cells were treated with the indicated doses of endostatin for 48 h and cell viability
determined using CCK-8 kit. (Data represent mean ± SD values of eight replicate experiments). (B)
Caspase 3, 9 levels in EOMA cells treated with saline, 100 nM endostatin, 100 μM CoCl2, 100 μM
CoCl2 plus 100 nM endostatin, detected by Western blotting. (C) Flow cytometry analysis of the
apoptosis of EOMA cell treated with saline, 100 nM endostatin, 100 μM CoCl2, 100 μM CoCl2 plus
100 nM endostatin by FITC Annexin V/Dead Cell Apoptosis Kit. The quantified results for apoptotic
cells is shown in panel D. (E) Cell-free culture media (Mock-untreated) 24 h later plus Endostatin
(Mock-untreated-Endostatin) or plus Endostatin (Mock-Endostatin) was detected by Western blot. (F)
Transwell migration assay using SVEC4-10 cells. Attractants include EOMA transfected with siRNA
that targeted the indicated genes, or with scrambled siRNA, and then treated with or without
endostatin. (G) The RNAi efficiencies were verified by qRT-PCR. Data are shown as mean ± SD. All
of the aforementioned experiments were repeated at least twice. (**, P<0.01; ***, P<0.005).
Supplementary Figure S3. The expression profiles of the endostatin-downregulated genes in
EOMA cells
EOMA cells were transfected with siRNA that targeted the indicated gene, or with scrambled siRNA.
The total RNA was extracted, reverse transcribed and the relative mRNA levels of the selected genes
detected by qRT-PCR. Data are shown as mean ± SD. All experiments were repeated at least twice
with three duplicates for each treatment. (*, P<0.05; **, P<0.01; ***, P<0.005).
Supplementary Figure S4. Endostatin inhibits the expression of CXCL1
(A) The relative cxcl1 mRNA level in EOMA cells transfected with siRNA targeting CDP, parp1 or
scrambled siRNA, and then treated with or without endostatin (30 nM), detected by qRT-PCR. (B)
CXCL1 level in supernatants from EOMA cells transfected with siRNA targeting aforementioned
genes and then treated with or without endostatin (30 nM), measured by sandwich ELISA. (C) The
RNAi efficiencies were verified by qRT-PCR. (D) EOMA cells were pretreated 100 nM endostatin,
or 5 μM Bay 11-7082, and then stimulated with TNF-α (10 ng/m) for 2 h, the relative mRNA level of
RelA/p65 were evaluated by qRT-PCR. (E) EOMA cells were pretreated 100 nM endostatin, or 5 μM
Bay 11-7082, stimulated with TNF-α (10 ng/m) for 2 h, and then treated with actinomycin D for
indicated time, the relative mRNA level of RelA/p65 were evaluated by qRT-PCR. (F) EOMA
transfected with pCMV6 empty vector (Mock) or pCMV6 vector encoding RelA/p65 (RelA/p65)
were treated with 100 nM endostatin for 24h. The amount of RelA/p65 in nuclear fraction was
detected by Western blot. Lamin B and tubulin served as markers for nuclear and cytoplasmic
proteins, respectively. The levels of indicated protein were quantified by ImageJ. Ratio means
RelA/p65/Lamin B. (G) The supernatants (Supplementary Fig. 4F) were collected and analyzed
using sandwich ELISA. Data are shown as mean ± SD. All of the aforementioned experiments were
repeated at least twice. (**, P<0.01; ***, P<0.005).
Supplementary Figure S5. CXCL1 is essential for the tumorigenesis of hemangioendothelioma
cells.
2×105 EOMA cells transfected with scrambled (N.C.), CXCL1 knockdown (Lenti-siCXCL1), or
CXCL1-overexpressing lenti-virus (Lenti-CXCL1) were subcutaneously inoculated into nude mice.
Fourteen days later mice were assigned randomly into two groups and given either 12 mg/kg
endostatin or placebo (saline) intratumorally every other day for 16 days. Sections of EOMA tumor
tissues were immunostained for PCNA (A) and cleaved caspase-3 (C), and the percentage of PCNA
positive cells (B) and cleaved caspase-3 positive cells (D) quantified. Values are expressed as mean ±
SD. (*, P<0.05; **, P<0.01).