Statistical every three days by a digital

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Statistical analyses were conducted with the PRISM Software (GraphPad
Software, San Diego, CA, USA) using two-tailed
Student’s t-test (two groups). Differences were considered statistically
significant at p value less than 0.05.
Data are expressed as mean ± SEM values from at least three independent
experiments.

Statistical
analysis

 

NMRI-Foxn1nu
/ Foxn1nu mice were purchased from JANVIER-LABS (Saint Berthevin,
France) and housed in clean specific pathogen free rooms. Care and use of the animals were in accordance with
institutional guidelines according to the Helsinki Declaration. MDA-MB-231
cells (5 × 106 cells in 200 µl PBS) were injected
subcutaneously into the right flank of each mouse (6 week old). Once the tumor
reached approximately 100-150 mm3, animals were administered
intraperitoneally every three days for 18 days with drugs. Tumors were measured every three days by a digital
caliper (Mitutoyo Corporation, Kawasaki, Japan).
Tumor volume was calculated using the formula V (mm3) = d1 × d22 ×
0.5, where d1 is the
length and d2 is the width
of the tumor. At the end of the treatment, the animals were sacrificed and
tumors were removed, weighed and in vivo
caspase-3 activity assay was performed as described in Supplementary Materials
and Methods.

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In vivo tumor model and treatment

 

Cancer cells were treated with BAPTA-AM (5 µM) or EGTA (100 µM) for 30
min then stimulated with or without simvastatin (100 nM) or doxorubicin (1 µM)
for 24 hours. Treated cells were washed and detached with trypsin-EDTA
solution. After which the cells were fixed with 4 % paraformaldehyde for 20 min
and permeabilized for 15 min in PBS containing 0.5 % Triton X-100. Cells were
stained with anti-BIM-phycoerythrin monoclonal antibody IgG1
(Santa Cruz Biotechnology, Inc., Heidelberg, Germany) at
4 °C for 30 min. As a negative control, cells were stained with
phycoerythrin-labeled isotype-matched antibodies. Data were analyzed by FlowJo
software (Tree Star Inc., Ashland, OR).

Measurement
of the Pro-apoptotic molecule BIM Level by Flow cytometry

 

Cancer cells were cultured on four well Lab-Tek® chamber slides (BD
Falcon, Le Pont de Claix, France), and exposed to Bapta-AM (5 µM) or EGTA (100
µM) for 30 min then stimulated with simvastatin (500 nM) or doxorubicin (5 µM)
for 3 hours. After which the cells were washed with
1X PBS solution. Slides were then fixed with 4 % paraformaldehyde for 30 min
and permeabilized for 20 min in PBS containing 0.5 % Triton X-100. Blocking was
20 min at room temperature in 10% boeuf serum albumin (BSA) in PBS, followed by
incubation with rabbit polyclonal anti-cleaved caspase-3 antibody (1:400, Cell
Signaling Technology) in 10 % BSA overnight at 4 °C. After washing with PBS,
cells were incubated with FITC-coupled anti-rabbit secondary antibody (1:300)
for 1 h at room temperature and rinsed in PBS. Finally, nuclei were stained
using DAPI (Sigma-Aldrich, St-Quentin Fallavier, France) at 1 ?g/mL. Images
were captured using a Leica
DMI6000 B inverted microscope.

Immunocytochemistry
studies of cleaved forms of caspase-3

 

Cytoplasmic Ca2+ monitoring was performed as previously
described47.

Live-cell
imaging of intracellular
Ca2+i levels

Hyaluronic acid (HA) hydrogels were synthesized at “Polymères, Biopolymères
Surfaces” laboratory (UMR 6270, University of Rouen, France) as previously described30-31. This
process has been described in two Europeans patents: “Improved Crosslinked
Hyaluronan Hydrogels for 3D culture” EP10305666. 9, June 22, 2010 and “Method
for Harvesting Cells Cultured in 3D Hydrogel Matrices” EP10305667.7, June 22,
2010. Before cell culture, hydrogels were cut into 2 × 2 × 0.5 mm cubes, sterilized
and then rehydrated in culture medium with 0.1 % antibiotics. Reticulated
HA hydrogel matrix was validated as a three-dimensional model in which tumor cells
can invade and grow30-31. Methods for
cells invasiveness and colony formation in HA hydrogels are
available in Supplementary Materials
and Methods.

Synthesis of HA hydrogels for 3D
culture, cells invasiveness and colony
formation

 

Human breast carcinoma specimens were obtained according to the
Declaration of Helsinki, and guidelines and protocols approved by the
Institutional Review Board of the Henri Becquerel Cancer Center (CLCC, Rouen,
France). All patients signed a written consent allowing the conservation and study
of their biological samples. Human breast specimens were obtained from 5 female
donors, ages 40, 47, 50, 51 and 58, and delivered in ice-cold culture medium to
the laboratory at Rouen University within 0.5-1 hour of sampling. Clinicopathological characteristics
of breast cancer patients enrolled were reported in supplementary Table 1. Cell culture media for human breast carcinoma cell
lines (MDA-MB-231 and MCF-7) were from Eurobio® (Courtaboeuf, France) and
Fura-2/AM from Abcam Biochemicals (Paris, France). All reagents are described
in Supplementary Materials and Methods.

Human
breast tumor, cell lines and reagents

Categories: Data

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