Cell viability assay, cytoplasmic and mitochondrial Ca2+
monitoring, 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.
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
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.
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.
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.
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:
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
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
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
Human breast tumor, cell lines and reagents