Statistical analyses were conducted with the PRISM Software (GraphPadSoftware, San Diego, CA, USA) using two-tailedStudent’s t-test (two groups). Differences were considered statisticallysignificant at p value less than 0.05.Data are expressed as mean ± SEM values from at least three independentexperiments.Statisticalanalysis The release of cell cytochrome c and apoptotic rate wereevaluated by Abcam’s ELISA and annexin V-FITC/PI kits (Paris, France) basicallyfollowing the manufacturer’s instructions. ELISAcytochrome 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 withinstitutional guidelines of the University of Rouen according to the HelsinkiDeclaration. MDA-MB-231 cells were harvested andresuspended in PBS, and 5 × 106 cells in a volume of 200 µl wereinjected subcutaneously into the right flank of each mouse. Once the tumorreached approximately 100-150 mm3, animals were randomized dividedinto six groups: Control (PBS); BAPTA-AM; doxorubicin (Doxo group); simvastatin(Simva group); BAPTA-AM and Doxo group; BAPTA-AM and Simva group, whichreceived BAPTA-AM (2 mg/kg), Doxo (4 mg/kg), Simva (5 mg/kg), with three tofour mice per group. The treatment was administered intraperitoneally everythree days for 18 days.
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Tumors were measured everythree 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 thelength and d2 is the widthof the tumor. At the end of the treatment, the animals were sacrificed andtumors were removed, weighed and in vivocaspase-3 activity assay was performed as described above.
In vivo tumor model and treatment Cells were stimulated with or without test compounds andthe pro-apoptotic molecule BIM level was evaluated by Flow cytometry38.Measurementof the Pro-apoptotic molecule BIM Level by Flow cytometry Cells were grown on the four well Lab-Tek® chamberslides (BD Falcon, Le Pont de Claix, France) before treating with Bapta-AM (5µM) 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 followinga method described earlier38. Cells imaging was captured with a Leica DMI6000B inverted microscope. Immunocytochemistrystudies of cleaved forms of caspase-3 Silencing experiments were performed with ON-Targetplus 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 DharmaFECTTranfection Reagent (Dharmacon) according to manufacturer’s instructions.Transfected cells were then assessed for Western blot and Ca2+ signalinganalysis.
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 laboratoryat Rouen University as previously described30-31. This process hasbeen described in two Europeans patents49, which validated thecross-linked HA hydrogel matrix as a three-dimensional model allowing thegrowth and invasion of tumor cells30-31. Hydrogel preparationmethods for cell culture were previously described49.Methods for cells invasivenessand colony formation in HA hydrogels weregiven in Supplementary Materials.Synthesis of HA hydrogels for 3Dculture, cells invasiveness and colonyformation Breast carcinoma samples were received from the Rouen HenriBecquerel Cancer Center (France) after approval of the protocols by its review committeein accordance with the Helsinki Declaration. Specimens were isolatedfrom five donors, and transferred in ice-cold RPMI 1640 medium to thelaboratory 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 theirsamples. Celllines (MDA-MB-231 and MCF-7) used in the study were obtained from EuropeanCollection of Authenticated Cell Cultures (ECACC, Porton Down, SP4 0JGSalisbury, UK). Details of reagents and antibodies were explained inSupplementary Materials. Human breast tumor, cell lines and reagents