Patients and tissues
All experiments using human
tissues
were performed
in
accordance with the Declaration of Helsinki and approved by the
Institutional Review Board of Fudan University. The clinical samples of
lung cancer patients were obtained from Fudan University Shanghai Cancer
Center (Shanghai, China). The tissues consisted of samples from 132
patients with NSCLC, including 100 cases of adenocarcinoma (ADC), 23
cases of squamous cell carcinoma (SCC), and 9 cases of other types of
NSCLC at different stages. On each tissue, healthy lung tissues were
also included.
Wound
healing assay
Wound healing was analysed using IBIDI
Culture-Inserts
(IBIDI GmbH, Martinsried, Germany). Cells were dissociated from plates
using 0.05% (w/v) trypsin and seeded into culture-insert plates at a
concentration of 2 × 104 cells per culture well. After
24 hours of incubation, culture inserts were removed. Photographs of the
movement of cells into the scratch area were taken after 24 h using a
light microscope.
Cell
migration
Cell migration was measured using transwell inserts (Corning Life
Sciences, Tewksbury, MA) according to manufacturer’s instructions.
Briefly, transwell chambers (8 mm pore size) were coated with 50 μL of
diluted matrigel. Cells suspended in serum-free medium at a density of
1.5×105 cells/mL were seeded (0.1 ml) in the upper
chambers and 0.5 mL medium containing 10% FBS was added to the lower
chambers, and PBS, 10 nmol/L thrombin, 25 nmol/L r-hirudin, 50 nmol/L
DTIP, 10 nmol/L thrombin + 25 nmol/L r-hirudin and 10 nmol/L thrombin +
50 nmol/L DTIP were
added
to both of the upper and lower chambers. After culturing for 24 hours,
cells were fixed in methanol and stained with 0.1% crystal violet. The
cells on the bottom of the filters were counted.
F-actin
staining assay and confocal microscopy
Cells were grown on 35-mm glass-bottom dishes and fixed with 4%
paraformaldehyde for 15 minutes and then permeabilized with 0.5% Triton
X-100 in PBS for 10 minutes. The cells were then blocked with 1% BSA
for 20 minutes. Cells were incubated with YF-488 phalloidin (US
Everbright®Inc) for 30 minutes and then stained with
DAPI for 5 minutes. The cells were observed under a confocal microscope.