This study using human cell lines was approved by the Ethics Committee for Animal Care and Use in Scientific Research from the Federal University of Rio de Janeiro. All cell culture reagents were purchased from Invitrogen (São Paulo, Brazil). The human mammary gland/breast epithelial cell line MDA-MB 231 was obtained from the American Type Culture Collection (ATCC® HTB-26™). Cells were routinely grown in RPMI medium containing 10 % fetal bovine serum (FBS), 1 % L-glutamine and 1 % penicillin-streptomycin, in a humidified 5 % CO2 atmosphere at 37 °C. Cells were cultured up to 70 %-100 % confluence and then some cultures were treated with the cholesterol depleting agent methyl-β-cyclodextrin (MbCD, Sigma, USA) at different concentrations for 2 h. After MbCD treatment, cultures were washed with fresh cultured medium (RPMI with 2 % FBS) and grown in culture medium with 2 % FBS for the next 2 to 48 h. Some cultures were grown in the presence of Wnt3a- or Wnt5a-enriched medium, which was obtained respectively from mouse Wnt3a or Wnt5a transfected L-cells. Other cells were grown in the presence of the Wnt activator 6-bromoindirubin-3'-oxime (Bio; Sigma) at a final concentration of 5 M or with the Wnt inhibitor Dkk1 (recombinant mouse Dkk1 protein; R&D Systems, USA) at a final concentration of 0.1 mg/ml.
Mouse L-cells culture and Wnt3a- and Wnt5a-conditioned medium preparation
L-Wnt3a (ATCC® CRL-2647™) and L-Wnt5a cells (ATCC® CRL-2814™) were cultured in DMEM supplemented with 10 % FBS and 0.4 mg/ml neomycin (Invitrogen) to maintain transgene expression during cell culture expansion. Conditioned medium from L-Wnt3a and from L-Wnt5a were collected as described below. Briefly, 1.3 x 106 cells were plated in 75 cm2 culture flasks with 14 ml of medium without antibiotics, and left to grow for four days. The first batch of medium was collected and replaced with 14 ml of fresh medium for another three days. The second batch of medium was then collected and the cells discarded. Both batches were mixed, sterile-filtered (0.22 μm) and stored at −20 °C for further assays.
Cell viability assay
Cell viability was determined using 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent (Sigma). Briefly, cells were plated at an initial density of 2.5 x 104 cells per well in 96-well plates and incubated for 24 h at 37 °C under 5 % CO2. Twenty-four-h cultures were treated with MbCD at a final concentration of 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 2, 5 or 10 mM for 2 h. Cells treated with medium only (RPMI medium containing 10 % FBS, 1 % L-glutamine and 1 % penicillin-streptomycin) served as a negative control group. After 24 or 48 h of treatment, the supernatant of each well was removed and cells were washed twice with PBS. Then, 10 μl of MTT solution (5 mg/ml in PBS) and 100 μl of medium were then added to each well and incubated for 4 h at 37 °C, 5 % CO2. The resultant formazan crystals were dissolved in dimethyl sulfoxide (100 μl) and absorbance intensities were measured in a microplate reader (FlexStation® 3 Reader, Molecular Devices, USA) at 570 nm. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated control cells.
Trypan blue exclusion assay
MbCD treated and non-treated cells were stained with 0.4 % trypan blue, and then examined microscopically to determine the total number of cells and trypan blue positive cells. The ratio of trypan blue positive cells was calculated as following: [(the number of trypan blue positive cells)/(the number of total cells)] × 100.
Plasma membrane integrity assay
To measure plasma membrane integrity, we assayed serum lactate dehydrogenase (LDH) levels and calculated percentages of LDH release to the medium. LDH activity was measured spectrophotometrically using a commercial kit (Doles, Goiás, Brazil). After MbCD treatment, 50 μl of the supernatant were transferred to an enzymatic assay plate and 50 μL of LDH substrate plus 5 μL ferric alumem were added and incubated for 3 min at 37 °C, protected from light. Then, 10 μl of NAD were added and absorbance intensities were measured in a microplate reader (FlexStation® 3 Reader, Molecular Devices, USA) at 490 nm. The percentage of LDH release was calculated by ([LDH] sample × 100)/total [LDH]. [LDH] sample was the LDH level of the sample (released in medium) and total [LDH] was the LDH content in the wells after addition of lysis solution (0.9 % Triton X-100).
Lipid extraction and cholesterol determination
The liquid culture media from untreated and MbCD treated cell cultures were collected after 2, 5, 8, 12, and 24 h after treatment for cholesterol determination. The extraction started by adding 4 ml of chloroform:methanol:HCl (2:1:0.075, v/v) and 1 ml of HCl 0.6 M to 1 ml the samples in glass tubes, followed by agitation and centrifugation (10 min, 300 x g). The organic phase (containing the lipids) was collected and 1 ml of chloroform:methanol:HCl 0,6 M (3:48:47; v/v) was added, followed by agitation and centrifugation (10 min, 300 x g). The final organic phase from each sample was dried under N2 gas and quantified gravimetrically. Cholesterol was determined according to the method described by Courchaine et al. . Briefly, the dried lipids from each sample in duplicate were solubilized in 750 μl of acetic acid, followed by the addition of 500 μl of a color reagent (0.05 g FeCl3, 2 ml H3PO4, 23 ml H2SO4), vigorously homogenized in a vortex mixer, and kept for 10 min at room temperature. The absorbance of each sample was determined in a spectrophotometer (FlexStation® 3 Reader, Molecular Devices, USA) at 550 nm. Cholesterol (Sigma) was used as the standard. The results are expressed as mg cholesterol per ml of sample. Aliquots (10 μl) from each sample were used for protein determination by BCA reagent (Thermo Scientific, USA), using bovine serum albumin as the standard.
Cell-based scratch assay
Cells were cultured in 24-well culture plates for 24 h up to 90 %-100 % confluence. Scratched wound lines on the upside of cultured cells were created by 200 μl yellow micropipette tip. The scratched cells were washed with PBS after removal of culture media. The cells were cultured for 2 h with 2 mM MbCD and after the removal of culture media cells were cultured for the next 2, 8, 12 or 24 h. All cell-based scratch assays were performed in the presence of the anti-mitotic reagent cytosine arabinoside (ara-c; Sigma) at a final concentration of 10−5 M in order to inhibit cell proliferation and analyze only cell migration. The wound area was measured from the image taken with an Axiovert 100 microscope (Carl Zeiss, Germany) by Image J program (NIH, USA) at 3 different sites from each wound area of gaps. Three independent experiments were performed.
Cells were rinsed with PBS and fixed with 4 % paraformaldehyde in PBS for 10 min at room temperature. Cells were then permeabilized with 0.5 % Triton-X 100 in PBS for 30 min. Cells were then washed in PBS for 30 min and incubated with Texas Red Phalloidin (Molecular Probes, USA) for 1 h at 37 °C. After incubation, cells were washed for 30 min with PBS. Nuclei were labeled with DAPI (4,6-Diamino-2-phenylindole dyhydrochloride; 0.1 μg/ml in 0.9 % NaCl) for 5 min. Cells were mounted in ProLong Gold antifade reagent (Molecular Probes) and examined with an Axiovert 100 microscope (Carl Zeiss, Germany). Images were acquired with an Olympus DP71 digital camera (Olympus, Japan).
Cell electroporation and luciferase assay
Electroporation was performed as previously described . Briefly, 2 x 106 MDA-MB 231 cells were ressuspended in 100 μl of electroporation buffer (5 mM KCl; 15 mM MgCl2; 120 mM Na2HPO4/NaH2PO4 pH 7.2; 25 mM Sodium Succinate; 25 mM Manitol) containing 4 μg of the reporter system plasmid 7xTcf-FFluc//SV40-PuroR (7TFP, Addgene plasmid 24308) to evaluate the activation of Wnt signaling  and 0.4 μg of TK-Renilla plasmid (Promega, USA). The cells were immediately transferred to a sterile 0.2 cm cuvette (Mirus Biotech®, USA) and electroporated using the program X-013 of the Lonza® Nucleofactor® II electroporation system. After electroporation, cells were plated in 12-well plates at an initial density of 105 cells/well and left to adhere overnight at 37 °C and 5 % CO2. On the following day, cultures were rinsed with PBS and incubated in triplicates with 2 mM MbCD, 10 % Wnt3a-medium or 5 μM Bio for 24 h. Cells were lysed with 80 μl of Reporter Lysis buffer/well (Promega, USA) and processed to a single freeze/thaw cycle at −80 °C to achieve complete lysis. After a brief centrifugation at 4 °C, supernatants were used to quantify luciferase activity through light detection by luminometer (Glomax, Promega, USA) after adding the Luciferase Assay Reagent according to the manufacture’s protocol.
The levels of IL-10 in cultured media were determined by enzyme-linked immunosorbent assay (ELISA) protocol as standardized by BD Biosciences (USA). Conditioned media were collected from untreated cultures and after 2, 8 and 24 h of treatment with 2 mM MbCD or 10 % Wnt3a or 10 % Wnt5a. Samples were centrifuged and the supernatants used for measurement of the concentration of IL-10. Briefly, the anti-mouse IL-10 capture monoclonal antibody (2 μg/ml, R&D Systems, USA) was absorbed on a polystyrene 96-well plate (Maxisorb, NUNC, Denmark), and the IL-10 present in the sample was bound to the antibody-coated wells. The recombinant mouse IL-10 (16 pg/ml to 0.125 pg/ml, R&D Systems, USA) was added to standard wells. The biotinylated anti-mouse IL-10 detecting mAb (0.1 μg/ml, R&D Systems, USA) was added to bind the IL-10 captured by the first antibody. After washing, streptavidin-horseradish peroxidase (streptavidin-HRP, Zymed, USA) was added to the wells to detect the biotinylated detecting antibody, and finally a Tetramethyl Benzidine chromogenic solution (TMB, Zymed) was added as a substrate, and a colored product formed in proportion to the amount of IL-10 present in the samples was stopped with HCl 1 N. Absorbance was read using 450 nm as wavelength. All samples were analyzed in duplicate. Results of cytokine concentration were expressed in pg/ml and were mean of three different experiments.
All the values were represented as the mean ± standard error. Statistical analysis was performed with one-way ANOVA on Ranks with Newman-Keuls Post Hoc test and statistical significance was defined as *p < 0.05 and ***p < 0.001.