Conceived and designed the experiments: JRM-C MDLM. Performed the experiments: C. Herencia JMM-M FC IE MB YA RS-M. Analyzed the data: C. Herencia JMM-M FC IE MB YA RS-M JRM-C. Contributed reagents/materials/analysis tools: C. Herrera JRM-C AR-A. Wrote the paper: JRM-C AR-A.
The authors have declared that no competing interests exist.
Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes
Wnt/β-catenin signaling pathway is a master regulator of cell fate and proliferation during embryonic development that plays a main role in the control of differentiation of embryonic and adult stem cells
Stem cells and cancer are inextricable linked and emerging data suggest an association between alterations in stem cells and the generation of cancer stem cells (CSC)
Human MSCs specific markers were evaluated by flow cytometry before and after 21 days of treatment with two protocols (CM1 and CM2) of hepatocytes differentiation. At 0 days, undifferentiated human bone marrow MSCs were negative for the expression of CD34, CD45, CD117, CD133, CD184 and VEGFR2, but positive for the expression of CD13, CD26, CD29, CD44, CD49e, CD90, CD105 and CD166 (
Levels of CD13, CD49e, CD166, CD133 and VEGFR2 in undifferentiated cells (UC), CM1 and CM2-treated cells after 21 days of culture. (Conditioned medium: CM). Values are expressed as mean of percentage ± standard deviation. (a p<0.001 and b p<0.01 vs. CM1-treated cells; +++ p<0,001 and + p<0,05 vs. undifferentiated cells).
Undifferentiatedcells (t = 0) | |
|
99.5±0.04 |
|
99.8±0.11 |
|
2.02±0.158 |
|
99.5±0.45 |
|
1.18±1.491 |
|
61.8±13.09 |
|
99.5±0.40 |
|
99.6±0.13 |
|
98.8±0.52 |
|
1.35±0.979 |
|
67.9±16.58 |
|
0.96±0.179 |
|
2.04±0.311 |
Data are included as mean ± standard deviation.
To compare the potential for hepatic differentiation of both protocols, the expression of hepatospecific genes was evaluated measuring mRNA levels by RT-PCR and protein expression by immunocytochemistry. The mRNA levels of albumin (ALB), α-fetoprotein (αFP), α1-antitrypsin (α1-AT), C/EBPα and CYP3A5 were strongly induced in human MSCs treated with CM1 or CM2 at 7, 14 and 21 days, compared to undifferentiated cells (UC). Comparatively, at 21 days of differentiation, there are no significant differences between both differentiation protocols. In CM2-treated cells there were not significant differences in the expression of these genes between cells to time 0 and cells after 48 h of treatment. The expressions of albumin and C/EBP were bigger in CM1 than CM2-treated cells while the expressions of α1-AT and CYP3A5 were increased in CM2 vs. CM1-tretaed cells. The expression of αFP was similar with both protocols (
Relative levels of mRNA expression of A) albumin (ALB), B) α-fetoprotein (αFP), C) α1-antitrypsin (α-1-AT), D) CCAAT/enhancer-binding protein beta (C/EBP) and E) cytochrome P450 (CYP3A5) were determined in human undifferentiated mesenchymal stem cells before and after differentiation with conditioned medium 1 (CM1) or 2 (CM2) after 7, 14 and 21 days of culture; Gene expression is shown as fold-changes compared to undifferentiated cells at each time. Values are expressed as mean ± standard deviation. All genes were increased significantly respect to undifferentiated cells (UC). a p<0.001, b p<0.01 vs. CM1 or CM2.
Expression of hepatospecific proteins, such as albumin, α1-antitrypsin, α-fetoprotein and cytokeratin 19 were also immunodetected in cells after 21 days of differentiation with both protocols CM1 or CM2 (
The presence of hepatospecific proteins such as albumin, α 1-antitrypsin, α-fetoprotein, cytokeratin-19 and PAS stain were evaluated by immunohistochemistry after 21 days of culture with conditioned medium CM1 or CM2. Arrows show positive staining area.
Since Wnt/β-catenin pathway plays a main role in the control of differentiation of adult stem cells, confocal microscopy was used to study the subcellular localization of β-catenin during differentiation into hepatocytes in both CM1 and CM2 (
A) To determine β-catenin subcellular localization, human mesenchymal stem cells undifferentiated (UC) and treated with conditioned medium 1 (CM1) or 2 (CM2) after 21 days of culture were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue). Merged image of β-catenin-FITC and DAPI staining is also shown. Original magnification: 40×. B) mRNA expression of Lrp5/6, Frizzled- 3 (FZD3) and c-myc was evaluated in undifferentiated cells and cells treated with conditioned medium 1 (CM1) or 2 (CM2). Fold of undifferentiated cells at 21 days of culture. a p<0.001 vs. CM1-treated cells. C)
To confirm Wnt/β-catenin pathway activation during CM2 protocol, the expression of several genes regulated by this pathway, such as Lrp5/6, Frizzled 3 and c-myc, were next analyzed. The differentiation of human MSCs into hepatocytes with CM2 increased the mRNA expression of Lrp5, Frizzled 3 and c-myc. Conversely, undifferentiated cells and CM1-treated cells showed much lower levels of expression of these genes (
A) Number of cells after 7, 14 and 21 days of culture in undifferentiated, CM1 and CM2-treated cells. Values expressed as mean ± standard deviation. a p<0.001 vs. CM1-treated cells and undifferentiated cells. B) The presence of nuclear PCNA (brown nucleus) was evaluated by immunohistochemistry after 21 days of culture in undifferentiated cells, CM and CM2-treated cells. Image is representative of three experiments. Original magnification: 20×. C) Cell cycle was analyzed at 21 days of hepatocyte differentiation in undifferentiated, CM1 and CM2-tretaed cells. Data are showed as mean of percentage plus standard deviation. a p<0.001 vs. CM1-treated cells, ++ p<0.01 and +++ p<0.001 vs. undifferentiated cells. D) Primary spheroid assay with count of number of cells after 4 days of culture with conditioned medium for spheroid formation. Data are showed that mean ± standard deviation (a p<0.001 vs. CM1-treated cells and +++ p<0.001 vs. undifferentiated cells). E) Secondary spheroid formation assay. Number of secondary spheroids was counted in an inverted microscope. Three experiments were carried out and data are expressed as mean ± standard deviation (a p<0.001 vs. CM1-treated cells and +++ p<0.001 vs. undifferentiated cells). F) Detail of secondary spheroids is showed in the microphotographs of undifferentiated cells, CM1 and CM2-treated cells.
Nuclear staining of PCNA was significantly higher in CM2-treated cells than in undifferentiated or CM1-treated cells (
For spheroid assay, differentiated cells for 21 days were cultured in low adherent plates for 4 days. Primary spheroids were detected in all groups although the number of spheres seemed be higher in CM2-treated cells. To quantify this data spheres were digested with trypsin-EDTA and subsequently counted. It is interesting to note that the capability to form spheres and the number of cells was higher in CM2-treated cells than the other cells (
A proteomic DIGE approach was used to analyze the repertoire of proteins differentially expressed in control cells and hepatocytes obtained with CM1 or CM2 differentiation protocols. The DIGE analysis showed 39 differentially expressed proteins, and 17 of them were identified, including chaperones, metabolic, structural, proteolytic and apoptosis-related proteins (
Relative abundance of specific proteins (DIGE analysis) in human mesenchymal stem cells undifferentiated after 21 days of culture (UC21d) and in mesenchymal stem cells differentiated into hepatocytes with conditioned medium 1 (CM1) or 2 (CM2). B) Western blot confirmation of the changes observed by DIGE analysis in the abundance of some proteins in CM1 and CM2 hepatocytes: Adenine phosphoriobosyl transferase (APT), cathepsin B precursor (CATB), L-lactate dehydrogenase β chain (LDHB), transgelin (TGL2), tropomyosin β chain (TPM2) and nuclear β-catenin. Tubulin and TFIIB were used as cytoplasm and nuclear loading control respectively.
Protein name | NCBI Acc n° | Sequenced Peptides | Sequence Cov (%) | Av. Ratio CM1/CM2 | p value | Subcellular location | Molecular function |
|
|||||||
Tropomyosin beta chain | P07951 | 10 | 33 | −2,68 | 0,0031 | Cyt | Muscle contraction |
Transgelin | Q01995 | 5 | 32 | −9,61 | 0,0024 | Cyt/Nuc | Muscle protein |
Collagen alpha-2 (VI) chain | P12110 | 3 | 2,9 | - | - | Secr | Extracellular matrix structural constituent |
|
|||||||
Heat-shock protein beta-1 | P04792 | 7 | 40 | - | - | Cyt/Nuc | Heat shock protein binding |
DnaJ homolog subfamily B member 11 precursor | Q9UBS4 | 1 | 17 | −4,82 | 0,0015 | ER | Heat shock protein binding |
Peptidyl-prolyl cis-trans isomerase A | P62937 | 2 | 17 | - | - | Cyt | Isomerase |
|
|||||||
Triosephosphate isomerase | P60174 | 11 | 56 | 1,45 | 0,0260 | Cyt | Isomerase |
Inorganic pyrophosphatase | Q15181 | 2 | 14 | 1,57 | 0,0270 | Cyt | Inorganic diphosphatase activity |
Adenine phosphoribosyltransferase | P07741 | 4 | 25 | 1,89 | 0,0027 | Cyt | AMP binding |
L-lactate dehydrogenase beta chain | P07195 | 5 | 29 | −9,64 | 0,0026 | Cyt | Oxidoreductase |
NADH ubiquinone oxidoreductase 30 KDa subunit | O75489 | 1 | 9 | - | - | Mit | NADH dehydrogenase (ubiquinone) activity |
Glutamate dehydrogenase 1 mitochondrial | P00367 | 1 | 8 | - | - | Mit | Oxidoreductase |
|
|||||||
Peroxirredoxin-4 | Q13162 | 4 | 16 | - | - | Cyt | Thioredoxin peroxidase activity |
Elongation factor 1-delta | P29692 | 4 | 24 | - | - | Cyt | Signal transducer activity |
Annexin A5 | P08758 | 22 | 73 | - | - | Cyt | Calcium-dependent phospholipid binding |
|
|||||||
Cathepsin B precursor | P07858 | 4 | 18 | 2,79 | 0,0140 | Lys | Hydrolase, protease, thiol proteose |
Cathepsin D precursor | P07339 | 7 | 29 | 1,57 | 0,0410 | Lys | Aspartyl protease, hydrolase |
Cyt: Cytoplasm; Nuc: Nucleus; Secr: Secreted protein; ER: Endoplasmic reticulum; Mit: Mitochondria; Lys: Lysosome.
Hepatocytes differentiation has been achieved using different types of stem cells, MSC
Wnt/β-catenin pathway activation took place in CM2-treated cells, with nuclear β-catenin translocation and up-regulation of genes related to this pathway. Treatment of cells with another protocol (CM1) also induced hepatic differentiation but without the concurrence activation of Wnt/β-catenin pathway. We show for the first time the capability of CM1 (HGF+FGF7) to differentiate human MSC into hepatocytes. Our results show also that differentiation into hepatocytes may be induced with or without activation of Wnt/β-catenin pathway. Our results with CM1-treated cells are consistent with other studies where down-regulation of Wnt/β-catenin pathway during hepatic differentiation is observed
Finally, our proteomic analysis showed a higher presence of hepatocellular carcinoma-related proteins, such as cathepsin β precursor, cathepsin D precursor, adenine phosphoribosyl transferase, L-lactate dehydrogenase, triosephosphate isomerase, inorganic pyrophosphatase or peptidyl prolyl cis-trans isomerase, in CM2 treated cells compared to CM1 treated cells. A high expression of these proteins has been observed in hepatic tumor and metastasis
In contrast, other proteins are down-regulated in tumoral processes, including hepatocellular carcinoma. Tropomyosin β chain, transgelin or annexin A5, with a lower expression in CM2- treated cells compared to CM1-treated cells, are down-regulated proteins in hepatocellular carcinoma. Tropomyosin plays a role of stabilization of actin filaments and in the suppression of cellular transformation in non muscle cells, such as hepatocytes
In summary, our study demonstrates that Wnt/β-catenin down-regulation is not necessary for hepatocyte differentiation of MSC. We show for the first time a cross-talk between human bone marrow MSC hepatocytes differentiation, Wnt/β-catenin pathway and a tumoral phenotype. The activation of Wnt/β-catenin during human MSC differentiation into hepatocytes is associated with abnormal proliferation, expression of CSC markers, spheroid formation and the generation of liver cells with tumoral characteristics, in contrast to hepatocytes differentiated without Wnt/β-catenin activation. Exploration of the differences between cancer stem cells from normal stem cells is crucial not only for the understanding of tumor biology but also for the prevention of potential complications derived from future liver therapies with human MSC.
This study was approved by the Reina Sofia University Hospital Review Board. The procedures followed were in accordance with the ethical standards of the ethic committee from Hospital Reina Sofía and with the Declaration of Helsinki. All samples were collected after written informed consent.
Human bone marrow (BM) was aspirated from the iliac crest of healthy donors. Fresh BM was cultured in flasks (FalconTM, BD Pharmigen, Franklin Lakes, NJ) seeding 10 µl BM cells/cm2 with alpha-minimum essential medium (α-MEM) supplemented with 2 mM L-glutamine, 15% fetal bovine serum (FBS) (BioWhittaker, Switzerland), 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 1 ng/ml of fibroblast growth factor-basic (FGF-b, Peprotech EC, London, UK)
Two different differentiation protocols were applied to confluent human MSCs for their differentiation into hepatocytes. An explicative diagram is included in Supporting Information (
For immunophenotype studies, basal and differentiated human MSC were detached and stained with fluorescein- or phycoerythrin-coupled antibodies and analyzed with a FACSCalibur flow cytometer (Becton, Dickinson). Anti-CD34-FITC, anti-CD45-PE and anti-CD133 were purchased from Miltenyi Biotec (Berlin, Germany), anti-CD73-PE, anti-CD90- PE and anti-CD166 were from BD Pharmigen (Franklin Lakes, NJ), anti-CD13-FITC, anti-CD44-FITC and anti-CD49e-FITC were from Beckman Coulter, Inc (CA, USA), anti-CD105-FITC was from R&D Systems (MN, USA), and anti-CD29-FITC, anti-CD184-PE and VEGFR2 were from eBioscience, Ltd (London, UK).
Total RNA was extracted following a modification of Chomezynski and Sacchi's protocol with Trizol reagent Sigma-Aldrich (St Louis, MO). Total RNA was quantified by spectrophotometry (ND-1000, Nanodrop Tecnologies, DE, USA). One µg of total RNA was treated with DNAse (DNAse kit, Sigma-Aldrich, St Louis, MO) and complementary DNA was amplified using the QuantiTect Reverse Transcripction kit (Qiagen, Hilden, Germany). Primers were designated with the free Oligo 7 software and their sequences are listed in
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Human MSCs were cultured on chamber slides (Nunc, Rochester, NY, USA) for 21 days and then were fixed and treated during 20 min with 0.01 M citrate buffer pH 6. Cells were incubated for 1 h at room temperature with: anti-PCNA (1∶75 dilution, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-albumin (DakoCytomation Glostrup, Denmark, 1∶2000 dilution), anti-α-fetoprotein (R&D Systems, Minneapolis, MN, USA, 10 µg/ml), anti-cytokeratin-19 (R&D Systems, Minneapolis, MN, USA, 10 µg/ml), or anti-α-1-antitrypsin (DakoCytomation Glostrup, Denmark, 1∶800 dilution) primary antibodies. HRP-labelled polymer conjugated to secondary antibodies was used for 30 minutes at room temperature and diaminobenzidine was added to detect positive staining. Finally, cells were counterstained with hematoxylin (DakoCytomation Glostrup, Denmark). During all the procedure three washes with PBS were performed after each step.
Undifferentiated and differentiated human MSCs were cultured on chamber slides and, after the corresponding treatments; they were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 minutes at room temperature. Samples were then treated with chilled methanol (−20°C) for 10 min and washed in PBS (3×, for 5 min) and then sequentially incubated for 60 minutes each with anti-β-catenin (1∶50, BD Pharmigen, Franklin Lakes, NJ) and anti-mouse IgG-FITC (DakoCytomation Glostrup, Denmark). Between incubations, slides were washed with PBS+1% BSA (Sigma-Aldrich) for 10 minutes. DAPI (Invitrogen, CA, USA) was used for nuclear stain. Cells were examined by confocal fluorescence microscopy using a confocal microscope (LSM 5 Exciter Carl Zeiss).
The number of undifferentiated cells, CM1 and CM2-treated cells was counted at 0, 7, 14 and 21 days of culture. Cells were treated with Trypsin-EDTA (Sigma), inactivated with medium plus FBS and washed with PBS. Trypan blue was used to measure the cellular viability and the count was carried out with a Neubauer chamber.
For cell cycle, the different types of cells (undifferentiated, CM1 and CM2-treated cells) were harvested after 21 days of differentiation. Cells were trypsinized and subsequently fixed in 70% cold ethanol overnight. After cells were centrifuged and washed with Hank's solution 1× (Sigma-Aldrich, St Louis, MO) twice. Cells were lysated with DNA extraction buffer which contained citric acid 0.1 M and anhydrous disodium phosphate 0.2 M (Sigma-Aldrich, St Louis, MO) for 5 minutes. After incubation, cells pellets were resuspended in 100 µl staining buffer which contining 50 µg/ml propidium iodine, 50 µg/ml RNase, 0.1% Triton-X-100 and 0.1 M EDTA in PBS (Sigma-Aldrich, St Louis, MO). Cells were incubated for 30 min in darkness. Finally, cells were resuspended in PBS and they were acquired at low speed using FACScaliber (Becton Dickinson, CA, USA). Cell cycle analysis was performed on FlowJo program based on the mathematical algorithm of Watson (Becton Dickinson, CA, USA).
To analyze the number of secondary spheroids undifferentiated cells, CM1 and CM2 treated cells were harvested at clonal dilution (cell/ul) on low adherence plates. After 4 days of culture the number of spheres was counted in an inverted microscope. Three experiments were carried out and the data are expressed as mean of number of spheres ± standard deviation. Representative microphotographs of secondary spheroids were taken in an inverted microscope to 10×.
To check 3-dimensional structure of spheroids, undifferentiated cells, CM1 and CM2-treated cells were collected from plates and stained with DAPI for 5 minutes. Subsequently cell were centrifuged gentlely and resuspended in 15 ul of PBS. Spheroids' mounting was carried out according to the protocol described by Weiswald et al
After acetone precipitation, protein samples (Control cells at 0 and 21 days of culture and hepatocytes obtained by CM1 or CM2) were solubilized in 2-D DIGE sample buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, buffered to pH 8. Protein concentration was determined using the Bradford's assay (Bio-Rad). Then, 50 µg protein was labelled with 400 pmol of CyDye DIGE Fluor minimal dyes (GE Healthcare) and incubated on ice in the dark for 30 min according to the manufacturer's instructions (Cy3, Cy5 for samples and Cy2 for internal control consisting of a mixture composed by equal amounts of protein from all samples). Paired samples were reverse-labeled in order to prevent potential dye labeling bias. The reaction was stopped by addition of 1 µl of 10 mM lysine and incubated on ice for 10 min. Samples were cup-loaded onto IPG strips, 24 cm, pH 3–11NL (GE Healthcare), and subjected to isoelectrofocusing (IEF) in IPGphor™ IEF System (GE Healthcare) according to the manufacturer's recommendations. Upon IEF, strips were incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, a trace of bromophenol blue), containing 0.5% DTT for 15 min and thereafter in the same buffer with 4.5% iodoacetamide for 15 min. For the second dimension, strips were loaded on top of 12.5% polyacrylamide gels and run (1 W/gel) for 12–14 h until the bromophenol blue dye reached the gel bottom-end. Subsequently, 2D gels were scanned using a TyphoonTM Trio Imager (GE Healthcare) at 100 µm resolution with λex/λem of 488/520, 532/580, and 633/670 nm for Cy2, Cy3, and Cy5 respectively. The photomultiplier tube was set to ensure that the maximum pixel intensity was between 90,000 and 99,000 pixels. Image analysis was performed using DeCyder 6.5 software (GE Healthcare) as described in the user's manual. Three independent experiments were performed for each experimental setup. Briefly, the differential in-gel analysis module was used for spot detection, spot volume quantification and volume ratio normalization of different samples in the same gel. Then the Biological Variation Analysis (BVA) module was used to match protein spots among different gels and to identify protein spots that exhibit significant differences. Manual editing was performed in the BVA module to ensure that spots were correctly matched between different gels, and to get rid of streaks and speckles. Differential expressed spots were considered for MS analysis when the fold change was larger than 1.2 and the p-value after T-test was below 0.05. Preparative gels were run with 350 µg of protein following the same procedure described above. Proteins were visualized by staining with SYPRO Ruby Protein Gel Stain (Bio-Rad) and images were acquired with a TyphoonTM Trio Imager using λex/λem of 532/560 nm. Spots differentially represented were excised manually and gel specimens were processed with a MassPrep station (Waters). In-gel tryptic digestion was performed with 12.5 ng/µl trypsin in 50 mM ammonium bicarbonate for 12 h at 37°C. The resulting peptides were extracted with 5% formic acid, 50% acetonitrile. Samples were then concentrated in a speed-vac before MS analysis.
Microcapillary reversed phase LC was performed with a CapLCTM (Waters) capillary system. Reversed phase separation of tryptic digests was performed with an Atlantis, C18, 3 µm, 75 µm×10 cm Nano EaseTM fused silica capillary column (Waters) equilibrated in 5% acetonitrile, 0.2% formic acid. After injection of 6 µl of sample, the column was washed during 5 min with the same buffer and the peptides were eluted using a linear gradient of 5–50% acetonitrile in 30 min at a constant flow rate of 0.2 µl/min. The column was coupled online to a Q-TOF Micro (Waters) using a PicoTip nanospray ionization source (Waters). The heated capillary temperature was 80°C and the spray voltage was 1.8–2.2 kV. MS/MS data were collected in an automated data-dependent mode. The three most intense ions in each survey scan were sequentially fragmented by CID using an isolation width of 2.5 and relative collision energy of 35%. Data processing was performed with MassLynx 4.0. Database searching was done with ProteinLynx Global Server 2.1 (Waters) and Phenyx 2.2 (GeneBio, Geneva, Switzerland) against Uniprot knowledgebase Release 12.3 consisting of UniprotKB/Swiss-Prot Release 54.3 and UniprotKB/TrEMBL Release 37.3 with 285.335 and 4.932.421 entries respectively. The search was enzymatically constrained for trypsin and allowed for one missed cleavage site. Further search parameters were as follows: no restriction on molecular weight and isoelectric point; fixed modification, carbamidomethylation of cysteine; variable modification, oxidation of methionine.
Cytosolic extracts were obtained with a lysis buffer A, pH 7.9, containing 10 mM Hepes, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 70 µg/ml Protease Inhibitor Cocktail, 0.5% Igepal CA-630 (Sigma-Aldrich, St Louis, MO). The suspension was centrifuged (13000 rpm, 3 min and 4°C) and supernatant was stored at −80°C until used. Nuclear extracts were obtained by incubating the pellet obtained as described above in a lysis buffer B, pH7.9, containing 20 mM Hepes, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 46 µg/ml Protease Inhibitor Cocktail (Sigma-Aldrich, St Louis, MO). Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories GmbH, Munich, Germany). For Western Blot analyses, equal amounts of protein were loaded and electrophoresed on 7% SDS-polyacrylamide gel (Invitrogen; CA, USA). The protein was transferred to a nitrocellulose membrane (Invitrogen; CA, USA), and blots were incubated in blocking solution (Bio-Rad Laboratories GmbH, Munich, Germany). Primary antibodies were diluted in TTBS+5% non fat dry milk powder. Anti-β-catenin antibody (Cell Signaling, Boston, MA, USA) was diluted 1∶1000 before use and anti-TFIIB (1∶500 dilution, Santa Cruz Biotechnology) was used as loading control of nuclear extract. Other primary antibodies used were: anti-p53 (1∶500) and anti-L-lactate dehydrogenase β chain (1∶200) from Santa Cruz Biotechnologies, anti-tropomyosin β chain (1∶500), adenine phosphoribosyltransferase (1∶500) and Transgelin (1∶2000) that were purchased from Novus Biologicals Littleton, CO, cathepsin B (4 µg/ml) from Sigma-Aldrich and tubulin 1∶10000 from Abcam (Cambridge, UK) were performed. Blots were immunolabeled using a horseradish peroxidase conjugated secondary antibody and developed on autoradiographic film using the ECL Plus Western Blotting Detection System from Amersham Biosciences U.K. Limited (Little Chalfont, England).
Data are expressed as mean ± SD. The difference between means from two different groups was evaluated by performing a t test and
(AVI)
(TIF)
(TIF)
We acknowledge the technical support provided by Esther Peralbo in performing the studies with Confocal Microscopy (IMIBIC).