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Methods
Materials and reagents
Cryptotanshinone was purchased from Xi’an Helin Biological Engineering Co.
Ltd (Xi’an, China). Gefitinib (G-4408) was obtained from LC Laboratories (Woburn,
MA). Lipopolysaccharide (LPS), 1-phenyl-2-thio-urea (PTU), tricaine (MS-222),
methylcellulose, collagen, Poly-L-lysine and bromodeoxyuridine (BrdU) were
obtained from Sigma-Aldrich (St. Louis, MO). DiI was purchased from Invitrogen
(Camarillo, CA, USA). VEGF165 was from Millipore (Millipore, Bedford, MA).
Actinomycin D was from Beyotime Biotechnology. Matrigel was purchased from BD
(BD Biosciences, San Diego, CA) and Mitomycin C was from Roche. The primary
antibodies used include: Dll4, Jagged1, Notch1 and Lamin A/C from Abcam (Abcam,
Cambridge, MA); TNFR1, TNFR2, NF-κB P65, p-NF-κB P65, p-IκB-α, STAT3,
p-STAT3, P38, p-p38, Akt and p-Akt from Cell Signaling Technology (Danvers, MA);
VEGF, TLR4 and IκB-α from Santa Cruz Biotechnology (Santa Cruz, CA). Erk,
p-Erk and GAPDH from Bioworld Technology and human TNF-alpha antibody was
purchased from R&D Systems (Minneapolis, MN)
Cell culture
HUVECs were purchased from Allcells LLC (Shanghai, China) and cultured in
0.5% gelatin-coated (Corning) 10-cm dishes supplemented with 10% fetal bovine
serum (Hyclone, Thermo scientific, US) and other supplements (Allcells LLC, cat. no.
H004, H004B). Cells were maintained in a humidified atmosphere (5% CO2/95% air)
at 37 °C and Passage 3–4 were used in this study.
Mouse melanoma cells (B16F10) and murine RAW264.7 monocyte macrophages
were obtained from American Type Culture Collection (Manassas, VA), and cultured
in DMEM medium (Gibco, US), supplemented with 10% FBS, 100 U/ ml penicillin,
and 100 mg/ml streptomycin in a humidified chamber at 37°C /5% CO2 and were
used not more than 15–20 passages after the initiation of cultures.
Conditioned media (CM) obtained from cancer cell supernatants was collected 1
day after B16F10 cells reached 70-80% confluence as described[1]. To examine the
effects of CT on cell function, cells at 80–90% confluence were treated with 0.5-8μM
of CT for indicated time before experiments.
Wound healing mobility assay
HUVECs were seeded into 6-well plates (5  105 cells/well) and allowed to grow
to confluency in M199 medium containing 20% FCS, growth factor and heparin. Cell
monolayer was then treated with mitomycin C (10μg/ml) for 20min, followed by
scratched with sterile P200 micropipette tips and washed three times with
phosphate-buffered saline (PBS) to remove cellular debris. Fresh medium with or
without LPS (1μg/ml) and various concentrations of CT were added into the wells at
37°C for 24h. To evaluate the migration abilities of HUVECs in different
experimental conditions, the wound area was measured with Image-pro Plus 6.0
software and expressed as the percentage of the closure area[2].
Transwell migration assay
Transwell migration was performed as previously described with minor
modifications[3]. Briefly, 2 × 105 cells/ml HUVECs treated by various concentrations
of CT with LPS (1μg/ml) or control (PBS) labeled with 10μM CMFDA in 250ul were
added to the upper chamber of Transwell Boyden Chamber (Cat. #3422, Corning
costar, US) with an 8μm pore polycarbonate filter, while the lower chamber contained
600μl M199 conditioned media containing 20% FBS as an inducer of cell migration.
After 6h of incubation at 37°C, non-migrating cells were removed from the top of
each filter by cotton swabs, then the cells that had migrated to the lower surface of
each filter were fixed with 4% PFA and were examined by fluorescent microscope.
Five random fields per filter were chosen and photographed and the number of cells
was manually counted.
ELISA assay
Human VEGF ELISA kit (Perpro Tech), human and mouse TNF-α-specific
ELISA kit (eBioscience) were used to determine the levels of VEGF and TNF-α in the
conditioned media or blood serum, respectively. Briefly, cell culture supernatants
from HUVECs at approximate 70-80% confluence treated with or without LPS and
CT were collected for the assay. The experiments were performed according to the
manufacturer’s instructions.
RNA extraction and Real-time PCR
Total RNA of HUVECs was isolated with Trizol. Total RNA (1μg) was used for
first-strand cDNA synthesis using Prime Script® RT reagent Kit with gDNA Eraser
system (Takara, Japan). The expression of TNF-α was analyzed by qPCR with Premix
Ex Taq™ (Probe qPCR) (Takara, Japan) using an Applied Biosystems® 7500
Real-Time PCR System. TNF-α was amplified using its specific primers (sense: 5’GCCTGCTGCACTTTGGAGT-3’, antisense: 5’- CTCGGGGTTCGAGAAGATG -3’)
as described. The mRNA values for each gene were normalized to internal control
GAPDH
mRNA
with
the
5’-CGAGATCCCTCCAAAATCAA-3’,
following
primers:
antisense:
(sense:
5’-
TTCACACCCATGACGAACAT-3’). The ratio of normalized mean value for each
treated group to vehicle control group (DMSO) was calculated.
Immunofluorescence staining
To determine the effects of CT on proliferation and intracellular NF-κB P65 or
HuR protein localization, HUVECs were directly seeded on coverslips coated with
polylysine (100μg/ml). HUVECs at 70–80% confluence treated with or without
various concentrations of CT, and BrdU (30μg/ml) in fresh media was analyzed by
BrdU incorporation assay as described[4]. For the protein distribution assay, cells
were starved with serum-free medium overnight. HUVECs were treated as indicated
and fixed on the slides by incubation of 4% paraformaldehyde for 1h at room
temperature. Thereafter, cells were permeabilized in PBS containing 0.1% Triton
X-100 for 10 min. Cells were blocked with 10% sheep serum at 37°C for 1h, followed
by incubated with indicated primary antibody (1:50) at 4°C overnight. Cells were
incubated with indicated FITC-conjugated secondary antibodies (1:200) along with 4',
6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. 5 random fields
were chosen and imaged by fluorescence confocal microscope (Olympus, Japan).
Western blot analysis
Total cell protein extracts were prepared with RIPA buffer containing protease
and phosphatase inhibitors[5]. Cytoplasmic and nuclear extracts were prepared using
the NE-PER® Nuclear and Cytoplasmic Extraction Reagents kit according to the
manufacturer’s instructions (Thermo, Rockford, US)[6]. Protein concentrations were
determined using the BCA Protein Assay Protein Assay kit (Pierce, US). Equal
amounts of cell lysates (25μg) were loaded on 10% SDS-PAGE and transferred onto
PVDF membranes. After membranes were blocked, they were incubated with specific
antibodies against indicated primary antibodies overnight at 4°C followed by
incubation with horseradish peroxidase-conjugated IgGs for 1 h at 37°C. Detection
was performed by the ECL system (Millipore, Braunschweig, Germany) and
visualized with the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
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