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Supplementary method section: Tsuchida R and Das B et al.
Side population (SP) analysis. The protocol was based on that described by Montanaro
et al (Montanaro et al., 2004). Briefly, 1-2 million cells in suspension (medium plus 10%
serum) were treated with Hoechst 33342 dye (4.5ug/ml) for 45 minutes at 370C with
intermittent mixing, with and without verapamil (50M). Following this incubation, cells
were washed with ice-cold PBS, and kept on ice. Propidium iodide was added (1g/ml),
and cells were analyzed for a SP fraction using fluorescence-activated cell sorting
(FACS). A Coulter Epics Flow cytometer was used, and the Hoechst dye was excited
with the UV laser at 351 to 364nm, and its fluorescence measured with a 515-nm side
population filter (Hoechst blue) and a 608 EFLP optical filter (Hoechst red). A 540 DSP
filter was used to separate the emission wavelengths.
Western Blot Analysis: The overall method has been previously described in detail (Das
et al., 2005). We used the following antibodies: phopho-ERK1/2, ERK1/2, phopho-AKT
and survivin (Cell-signaling, USA), Flt1 (Chemicon International, CA, USA), and
phospho-tyrosine kinase (Santa-Cruz Biotechnology, CA, USA). -tubulin (Neomarkers,
Fremont, CA), -actin and vinculin (Santa-Cruz Biotechnology, CA, USA) were used as
loading controls. Immunoblotting was detected by enhanced chemiluminescence (ECL;
Amersham Bioscience, Buckinghamshire, UK) according to the manufacturer’s
instructions.
Immunoprecipitation: We used a crude membrane preparation technique to detect the
phosphorylated form of Flt1 membrane receptor (Das et al., 2005). A total of g of
the membrane protein was immunoprecipitated using a phosphotyrosine antibody (Santa
Cruz) as previously described (Das et al., 2005). Immunocomplexes were recovered by
centrifugation, pellets were washed in solubilization buffer, boiled and resolved on a 10%
SDS-PAGE gel. Western blot was performed as described above, and membranes were
probed with a Flt1 monoclonal antibody.
Reverse transcriptase (RT) PCR: PCR conditions for all genes was 94 ℃ for 10
minutes for denaturing, followed by 35 cycles for 1 minute at 94 ℃, 56 ℃ for annealing
and 72 ℃ for extension. Primer used were: VEGF: Sense: 5'-CGAAGTG GTGA
AGTTCATGGATG-3'; Anti-sense: 5'-TTCTGTATCAGTCTTTCCTGGTGAG-3'. Flt1:
Sense: 5'-AAATAAGCACACCACGC-3‘; anti-sense: 5'-ACCTGCT GTTTTCGAT
GTTTC-3'. BCRP-1 5’-CCAGTTCCATGGCACTGGCCATA-3’; reverse: 5’-CAGGG
CCAC ATGA TT CTTCCACA-3’; GAPDH: 5’-AACGGGG AAGCT CACTG
GCATG-3’, reverse:5’-TCCACCACCCTGTTGCTGTAG-3’
Real-Time qantitative RT-PCR (qPCR). Real-time quantitative PCR was performed
using TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, USA) at 40
cycles with 100 ng of starting cDNA. RNA was quantified with the ∆∆Ct method as
described (Livak and Schmittgen, 2001) using SDS software version 2.2.1 (Applied
Biosystems, Foster City, USA).Amplification reactions were performed as specified by
the manufacturer using TaqMan® Universal Master Mix without AmpErase® UNG and
a 7900 Real Time PCR System (Applied Biosystems, Foster City, USA). RNA was
quantified with the ∆∆Ct method as described (Livak and Schmittgen, 2001) using SDS
software version 2.2.1 (Applied Biosystems, Foster City, USA). RNA levels were
normalized to GAPDH as an endogenous control. Each reaction was performed
in
quadrireplicates. TaqMan primer used in this study and their respective assay
identification number in the Applied Biosystem catalogue were: Oct-4 (NM_002701.4),
nanog (NM_024865), Flt1 (NM_002019.2), VEGF ( applied biosystem catalogue
number: Hs 00900054; 7 refseqs including NM_003376.4) BCRP1( NM_004827) and
GAPDH (both human and mouse).
Small Interfering RNA (siRNA): All siRNA reagents were obtained from Dharmacon
Research Inc, Chicago, USA. Flt1 siRNA SMART-pool-R reagent was used to
knockdown Flt1 expression in HOS cells. In vitro transfection was performed with
Lipofectamine-2000 (Invitrogen). Non-targeting siRNAs, pool (siCONTROL) were used
as a siRNA control. Cells were grown to 30-50% confluence in 24-well plates and treated
with 100 nM siRNA, according to manufacturer’s instructions, and experiments were
conducted 24hrs after transfection.
ELISA analysis: The measurements of VEGF was carried out using an ELISA kit
(R&D) according to the manufacturer’s protocols. Samples consisted of conditioned
media harvested after treatment with or without CDDP (2M) for 3 days in serum free
media. Fluorescence activity was converted to actual concentration by a standard curve
and divided by cell number.
Immunofluorescence and Immunohistochemistry: For the immunofluorescence
detection, the cells grown on coverslips were fixed with 4% paraformaldehyde for 10-20
minutes, permeabilized in 0.3% Triton-x100 in 10% goat serum for 1hr, and then
incubated overnight with primary antibodies, and then appropriate secondary antibody
was used. Images were obtained using confocal microscopy. Antibodies that were used in
this study included Flt1
Immunohistochemistry of
and phospho-ERK1,2 (Cell Signaling Inc, USA).
nanog and CD-34(Chemicon International, Millipore,
Billerica, MA) were performed in paraffin section according to standard procedure.
In vivo tumorigenicity assay: Tumorigenicity of the HOS cells was measured by tumor
incidence (number of tumors/number of injection of 2-10 million cells), and latency as
time required to form palpable tumors of more than 0.05 cc. Briefly, HOS tumor cells
were treated with 2M CDDP (with and without siRNA Flt1 treatment) for 3 days, and
then 2 million viable cells mixed with matrigel (100µl of matrigel mixed with 100µl of
cell suspension) were injected subcutaneously into both flanks of female nude mice
(Balb/c, nude/nude). Tumor size was measured with calipers on a weekly basis as
described (Patrawala et al., 2005). 4-6 mice were used for each set of experiments.
Das B, Yeger H, Tsuchida R, Torkin R, Gee MF, Thorner PS et al (2005). A hypoxiadriven vascular endothelial growth factor/Flt1 autocrine loop interacts with hypoxiainducible factor-1alpha through mitogen-activated protein kinase/extracellular signalregulated kinase 1/2 pathway in neuroblastoma. Cancer Res 65: 7267-75.
Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-8.
Montanaro F, Liadaki K, Schienda J, Flint A, Gussoni E, Kunkel LM (2004).
Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation
parameters. Exp Cell Res 298: 144-54.
Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005).
Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and
ABCG2- cancer cells are similarly tumorigenic. Cancer Res 65: 6207-19.