cell cycle examination, mitochondrial membrane
potential assay, ROS determination, caspase-3 activity measurement, and
immunoblots were all done as described in our previous work50 with some
modifications explained in the supplemental methods. Cell viability assay,
cytoplasmic and mitochondrial Ca2+ monitoring,

Additional
Methods

 

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.

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Statistical
analysis

 

The release of cell cytochrome c and apoptotic rate were evaluated by
Abcam’s ELISA and annexin V-FITC/PI kits (Paris, France) basically following
the manufacturer’s instructions.

ELISA
cytochrome c and apoptosis assays

 

Six week old male
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 of the University of Rouen according to the Helsinki Declaration.
MDA-MB-231 cells were harvested and resuspended in PBS, and 5 × 106
cells in a volume of 200 µl were injected subcutaneously into the right flank
of each mouse. Once the tumor reached approximately 100-150 mm3,
animals were randomized divided into six groups: Control (PBS); BAPTA-AM;
doxorubicin (Doxo group); simvastatin (Simva group); BAPTA-AM and Doxo group;
BAPTA-AM and Simva group, which received BAPTA-AM (2 mg/kg), Doxo (4 mg/kg),
Simva (5 mg/kg), with three to four mice per group. The treatment was
administered intraperitoneally every three days for 18 days. 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 before50.

In vivo tumor model and treatment

 

Cells were stimulated with or without test compounds and the
pro-apoptotic molecule BIM level was evaluated by Flow cytometry38.

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

 

Cells were grown on the four well Lab-Tek® chamber slides (BD Falcon, Le
Pont de Claix, France) before treating with Bapta-AM (5 µM) or EGTA (100 µM)
for 30 min, followed by 3 hours stimulation with simvastatin (500 nM) or
doxorubicin (5 µM). Immunocytochemistry analysis was carried out following a
method described earlier38. Cells imaging was captured with a Leica DMI6000 B inverted microscope.

Immunocytochemistry
studies of cleaved forms of caspase-3

 

Silencing experiments were performed with ON-Target plus Human siRNA (25
nM) targeted to STIM1, TRPC1, TRPC3, or Non-targeting siRNA (GE Healthcare Dharmacon, Lafayette, CO 80026, USA). Small interference RNAs
were introduced
into the cell by using DharmaFECT Tranfection Reagent (Dharmacon) according to
manufacturer’s instructions. Transfected cells were then assessed for Western
blot and Ca2+ signaling analysis. The small interference RNA sequences
used in the study were: Non-targeting (Control siRNA: UGGUUUACAUGUCGACUAA),
STIM1-targeting (STIM1 siRNA: GGUGGUGUCUAUCGUUAUU), TRPC1-targeting (TRPC1
siRNA: GGACUACGGUUGUCAGAAA), TRPC3-targeting (TRPC3 siRNA: GGAAGGACCUAGGGAAUAC).

Small
interference RNAs targeted to STIM1, TRPC1 and TRPC3

 

HA hydrogels were obtained from UMR 6270 PBS laboratory at Rouen University
as previously described30-31. This process has been described in two
Europeans patents49, which validated the cross-linked HA hydrogel
matrix as a three-dimensional model allowing the growth and invasion of tumor
cells30-31. Hydrogel preparation methods for cell culture were previously
described49. Methods for
cells invasiveness and colony formation in HA hydrogels were given in Supplementary Materials.

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

 

Breast carcinoma samples were received from the Rouen Henri Becquerel
Cancer Center (France) after approval of the protocols by its review committee
in accordance with the Helsinki Declaration. Specimens were isolated from five
donors, and transferred in ice-cold RPMI 1640 medium to the laboratory at Rouen
University within 0.5 to 1 hour. All subjects
(supplementary Table 1) associated with this study have signed a written
consent for the use of their samples. Cell lines (MDA-MB-231 and MCF-7) used in the study were obtained from
European Collection of Authenticated Cell Cultures (ECACC, Porton Down, SP4 0JG
Salisbury, UK). Details of reagents and antibodies were explained in
Supplementary Materials.

Human
breast tumor, cell lines and reagents

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