The authors have declared that no competing interests exist.
Conceived and designed the experiments: AZBA MB AFPL. Performed the experiments: AZBA MB FS MZ SY RRD BP RK MA AFPL. Analyzed the data: AZBA MB AG AFPL. Contributed reagents/materials/analysis tools: MA EG RDC AFPL. Wrote the paper: EG RDC JF AFPL.
The extracellular milieu is comprised in part by products of cellular secretion and cell surface shedding. The presence of such molecules of the sheddome and secretome in the context of the extracellular milieu may have important clinical implications. In cancer they have been hypothesized to play a role in tumor growth and metastasis. The objective of this study was to evaluate whether the sheddome/secretome from two cell lines could be correlated with their potential for tumor development. Two epithelial cell lines, HaCaT and SCC-9, were chosen based on their differing abilities to form tumors in animal models of tumorigenesis. These cell lines when stimulated with phorbol-ester (PMA) showed different characteristics as assessed by cell migration, adhesion and higher gelatinase activity. Proteomic analysis of the media from these treated cells identified interesting, functionally relevant differences in their sheddome/secretome. Among the shed proteins, soluble syndecan-1 was found only in media from stimulated tumorigenic cells (SCC-9) and its fragments were observed in higher amount in the stimulated tumorigenic cells than stimulated non-tumorigenic cells (HaCaT). The increase in soluble syndecan-1 was associated with a decrease in membrane-bound syndecan-1 of SCC-9 cells after PMA stimuli. To support a functional role for soluble syndecan-1 fragments we demonstrated that the synthetic syndecan-1 peptide was able to induce cell migration in both cell lines. Taken together, these results suggested that PMA stimulation alters the sheddome/secretome of the tumorigenic cell line SCC-9 and one such component, the syndecan-1 peptide identified in this study, was revealed to promote migration in these epithelial cell lines.
Oral cancer is one of the most common malignancies worldwide and despite improvements in diagnosis and treatment, the overall survival rate for advanced patients has not been significantly improved over the last three decades
New approaches on clinical proteomics, such as secretome-based analysis, have been developed to identify novel biomarkers. Secretome/sheddome is a proteomic area that allows the analysis of a dynamic extracellular environment including secreted, released, degraded or shed proteins
In order to evaluate the differences between the secretome/sheddome of normal and tumorigenic cells, two epithelial cell lines, HaCaT and SCC-9, were treated with phorbol-ester (PMA). Here we showed that PMA stimulation induced distinct migration, adhesion and gelatinase activity as well as differences in the secretome/sheddome. Components in the media such as soluble and fragments of syndecan-1 were found mainly in stimulated tumorigenic cells. Syndecans are known family of cell surface proteoglycans that play regulatory roles in many biological processes, including migration, proliferation, wound healing, inflammation, angiogenesis and tumorigenesis
Fifty-three proteins were identified in the extracellular media and classified as extracellular matrix proteins, secreted proteins, membrane-bound proteins, and intracellular proteins that have a membrane projection. Differences between the cells either treated or not with PMA were observed, and based on the ratio of quantitative values, proteins with changes higher than 1.5-fold (i.e. >1.5 or <0.66) were considered as significantly regulated by PMA treatment (
Accession Number | Protein Identification | HaCat + PMA/HaCaT | SCC-9+ PMA/SCC-9 | SCC-9/HaCaT | SCC-9+PMA/ HaCaT+PMA |
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IPI00374563 | Agrin | 7.88 | absent | only in HaCaT | only in HaCat+PMA |
IPI00022418 | Isoform 1 of Fibronectin | absent | only in SCC+PMA | absent | only in SCC+PMA |
IPI00003951 | Isoform 1 of Laminin subunit alpha-3 | 5.09 | 14.17 | 0.46 | 1.29 |
IPI00015117 | Isoform Long of Laminin subunit gamma-2 | only in HaCaT + PMA | only in SCC+PMA | absent | 0.70 |
IPI00783665 | Laminin subunit alpha-5 | only in HaCaT + PMA | absent | absent | only in HaCat+PMA |
IPI00299404 | Laminin subunit beta-3 | 2.36 | Only in SCC+PMA | only in HaCaT | 0.39 |
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IPI00011229 | Cathepsin D | 1.28 | 1.30 | 0.67 | 0.68 |
IPI00297487 | Cathepsin H | 1.54 | absent | only in HaCaT | only in HaCat+PMA |
IPI00021738 | Collagenase 3 (MMP-13) | only in HaCaT + PMA | absent | absent | only in HaCat+PMA |
IPI00023673 | Galectin-3-binding protein | absent | only in SCC | only in SCC | absent |
IPI00023728 | Gamma-glutamyl hydrolase | only in HaCaT | absent | only in HaCaT | absent |
IPI00008561 | Interstitial collagenase | only in HaCaT + PMA | absent | absent | only in HaCat+PMA |
IPI00026314 | Isoform 1 of Gelsolin | only in HaCaT | absent | only in HaCaT | absent |
IPI00291262 | Isoform 1 of Clusterin | only in HaCaT | absent | only in HaCaT | absent |
IPI00029658 | Isoform 1 of EGF-containing fibulin-like extracellular matrix protein 1 | only in HaCaT + PMA | absent | absent | only in HaCat+PMA |
IPI00387168 | Isoform 1 of Proprotein convertase subtilisin/kexin type 9 | 1.57 | absent | only in HaCaT | only in HaCat+PMA |
IPI00783625 | Isoform 1 of Serpin B5 | 3.03 | absent | only in HaCaT | only in HaCat+PMA |
IPI00013890 | Isoform 1 of 14-3-3 protein sigma | 0.59 | absent | only in HaCaT | only in HaCat+PMA |
IPI00480121 | Kallikrein-10 | only in HaCaT + PMA | only in SCC+PMA | absent | 10.43 |
IPI00293276 | Macrophage migration inhibitory factor | 0.16 | 1.12 | 0.86 | 5.97 |
IPI00216691 | Profilin-1 | absent | 0.84 | only in SCC | only in SCC+PMA |
IPI00013895 | Protein S100-A11 | only in HaCaT | absent | only in HaCaT | absent |
IPI00294879 | Ran GTPase-activating protein 1 | only in HaCaT | absent | only in HaCaT | absent |
IPI00009342 | Ras GTPase-activating-like protein IQGAP1 | 0.34 | absent | only in HaCaT | only in HaCat+PMA |
IPI00296099 | Thrombospondin-1 | 3.09 | only in SCC+PMA | only in HaCaT | 0.20 |
IPI00018219 | Transforming growth factor-beta-induced protein ig-h3 | 0.95 | 1.00 | 0.39 | 0.41 |
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IPI00218918 | Annexin A1 | 0.96 | 2.19 | 0.85 | 1.93 |
IPI00024095 | Annexin A3 | absent | only in SCC | only in SCC | absent |
IPI00329801 | Annexin A5 | absent | 8.06 | only in SCC | only in SCC+PMA |
IPI00414320 | cDNA FLJ55482. highly similar to Annexin A11 | Only in HaCaT | absent | only in HaCaT | absent |
IPI00015688 | Glypican-1 | 2.07 | absent | only in HaCaT | only in HaCat+PMA |
IPI00010271 | Isoform A of Ras-related C3 botulinum toxin substrate 1 | absent | only in SCC | only in SCC | absent |
IPI00218474 | Isoform 1 of Beta-enolase | 4.12 | 0.07 | 74.08 | 1.25 |
IPI00465248 | Isoform alpha-enolase of Alpha-enolase | 1.12 | 0.28 | 7.44 | 1.86 |
IPI00031030 | Isoform 1 of Amyloid-like protein 2 | only in HaCaT + PMA | absent | absent | only in HaCat+PMA |
IPI00418169 | Isoform 2 of Annexin A2 | 1.04 | 0.85 | 0.92 | 0.75 |
IPI00871158 | Isoform 2 of Annexin A8-like protein 2 | absent | only in SCC | only in SCC | absent |
IPI00297160 | Isoform 12 of CD44 antigen | 3.18 | only in SCC+PMA | only in HaCaT | 1.31 |
IPI00027493 | Isoform 2 of 4F2 cell-surface antigen heavy chain | 0.23 | 3.46 | 1.58 | 24.32 |
IPI00554711 | Junction plakoglobin | 1.53 | absent | only in HaCaT | only in HaCat+PMA |
IPI00026952 | Plakophilin-3 | 3.05 | only in SCC | 1.18 | only in HaCat+PMA |
IPI00002441 | Syndecan-1 | absent | only in SCC+PMA | absent | only in SCC+PMA |
IPI00011564 | Syndecan-4 | 1.01 | only in SCC+PMA | only in HaCaT | 8.18 |
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IPI00020599 | Calreticulin | absent | only in SCC | only in SCC | absent |
IPI00298237 | cDNA FLJ56402. highly similar to Tripeptidyl-peptidase 1 | 1.81 | absent | only in HaCaT | only in HaCat+PMA |
IPI00843975 | Ezrin | 0.64 | 2.27 | 0.34 | 1.19 |
IPI00163187 | Fascin | 1.22 | 2.21 | 0.39 | 0.71 |
IPI00024067 | Isoform 1 of Clathrin heavy chain 1 | absent | 2.53 | only in SCC | only in SCC+PMA |
IPI00014898 | Isoform 1 of Plectin-1 | absent | only in SCC | only in SCC | absent |
IPI00291175 | Isoform 1 of Vinculin | only in HaCaT + PMA | absent | absent | only in HaCat+PMA |
IPI00219365 | Moesin | only in HaCaT + PMA | only in SCC+PMA | absent | 1.01 |
IPI00915869 | Putative uncharacterized protein MDH1 | absent | only in SCC | only in SCC | absent |
IPI00298994 | Talin-1 | Only in HaCaT | 1.53 | 24.56 | only in SCC+PMA |
The HaCaT and SCC-9 cells were treated with PMA for 24 h, the media were collected, the proteins were digested with trypsin and analyzed by LC-MS/MS. The data were submitted to Mascot search engine and the .dat files from Mascot output were analyzed in Scaffold Q+, which calculates the quantitative value by normalizing spectral counts across the experiments. The ratio of quantitative value of the PMA-stimulated cells/DMSO-treated cells for each protein is shown in the table. The proteins exclusively found in one condition are indicated as “only” and the proteins that are not present are indicated as “absent”.
Non-tumorigenic cells stimulated with PMA showed exclusively 14 up-regulated proteins, including agrin, laminin subunit alpha-5, cathepsin H, collagenase 3, interstitial collagenase, EGF-containing fibulin-like extracellular matrix protein 1, proprotein convertase subtilisin, serpin B5, amyloid-like protein 2, glypican-1, isoform 1 of beta-enolase, junction plakoglobin, plakophilin-3, tripeptidyl-peptidase 1 and vinculin. Tumorigenic cells also showed exclusively up-regulated proteins, such as fibronectin, annexin A1, annexin A5, 4F2 cell-surface antigen heavy chain, syndecan-1, syndecan-4, ezrin, fascin, clathrin and talin-1. However, there were commonly up-regulated proteins in response to PMA activation, namely laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, kallikrein-10, thrombospondin-1, CD44 antigen and moesin.
The analysis of peptides in the media showed that the treatment with PMA induced the increase of syndecan-1 fragments (
Endogenous peptides were identified by LC-MS/MS in the media after 24 h of PMA-treatment. The spectrum of syndecan-1 peptide (m/z 1019.5598, +2) was manually validated for b and y ion series.
Accession number | Protein Identification | Experiment 1 | Experiment 2 | ||||||||||||||
HaCaT | HaCaT+ PMA | SCC-9 | SCC- 9+PMA | HaCaT | HaCaT+ PMA | SCC-9 | SCC-9+ PMA | ||||||||||
Run 1 | Run 2 | Run 1 | Run 2 | Run 1 | Run 2 | Run 1 | Run 2 | Run 1 | Run 2 | Run 1 | Run 2 | Run 1 | Run 2 | Run 1 | Run 2 | ||
IPI00002441 |
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SQGLLDRKEVLGGVIAGGLVG | 3 | 4 | 6 | 6 | 4 | 5 | 8 | 9 | 3 | 2 | 3 | 6 | 8 | 7 | |||
SQGLLDRKEVLGGVIAGGLVGLI | 2 | 2 | 3 | 3 | 3 | 4 | 5 | 4 | 5 | 1 | 3 | 3 | |||||
SQGLLDRKEVLGGVIA | 1 | 2 | 5 | 5 | |||||||||||||
LDRKEVLGGVIAGG | 2 | 1 | |||||||||||||||
SQGLLDRKEVLGGVIAGG | 1 | ||||||||||||||||
RNQSPVDQGATGASQGLLDRKE | 2 | 2 | 2 | ||||||||||||||
VLGGVIAGGLVG | |||||||||||||||||
DLHTPHTED | 1 |
The HaCaT and SCC-9 cells were treated with PMA for 24 h, the media were collected and the endogenous peptides were analyzed by LC-MS/MS. The total numbers of the unique peptides and the number of spectral counts are shown in bold and the number of spectral counts is shown for each sequence. There is a statistically significant difference in the number of spectral counts of syndecan-1 fragments between SCC-9 cells and SCC-9 cells treated with PMA (Mann-Whitney test, p = 0.0273).
The effect of PMA on migration was evaluated by scratch assays (
PMA and vehicle effect on the migration of HaCaT
HaCaT (p = 0.002, Students'
Adhesion assay showed that the cells stimulated by PMA decreased the ability to adhere to extracellular matrix proteins. HaCaT (A) and SCC-9 (B) cells had the ability to adhere in Matrigel™ diminished after 24 h of PMA treatment. Three independent experiments were performed with triplicates. Columns represent mean ± SD (n = 3) and * indicates p<0.01, normalized with the control (vehicle: DMSO). (C) Representative micrographs (magnification 40×) of adherent cells after PMA and vehicle (DMSO) treatments.
HaCaT and SCC-9 cells were treated with PMA and after 24 h the gelatinase activity was evaluated in the conditioned media. There was an increase of activity upon PMA treatment in both cell lines for ∼72 kDa gelatinase (p = 0.01 and p = 0.02 for HaCaT and SCC-9 cells, respectively, Students'
Representative 1D-zymography of conditioned media (12 µg of proteins) of HaCaT and SCC-9 cells treated or not with 50 ng/ml PMA two times with 12 h interval, collected after 24 h. The gelatinase activity of immunoprecipitated MMP-2 was used as positive control. Numbers on the left indicate the molecular mass marker mobility (A). The densitometry of clear areas showed the increase of activity after PMA treatment (B). Columns represent mean ± SD (n = 3) and * indicates p<0.05.
The loss of membrane-bound syndecan-1 in SCC-9 cells was confirmed by immunofluorescence in a time-course experiment performed after 5 min, 30 min and 24 h upon PMA treatment (
SCC-9 cells were treated with PMA for 5 min, 30 min and 24 h, fixed, and labeled for syndecan-1 with goat anti-syndecan-1 antibody. (A) Immunofluorescence images revealed diminished syndecan-1 in membrane localization after 30 min and 24 h of PMA treatment. Green: anti-goat antibody conjugated with Alexa Fluor 488. Blue: DAPI. (B) Example of masks used for quantification of syndecan-1 in the cell surface membrane. (C) Quantification performed by the Operetta high content image system showed loss of cell membrane of syndecan-1 after 30 min and 24 h of PMA treatment. Three independent experiments were performed. Columns represent mean ± SD (n = 3) and * and ** indicate p<0.05 and p<0.01, respectively.
The synthetic peptide of syndecan-1 (SYN-1) and its scrambled were evaluated at the concentrations of 1 µM, 10 µM and 100 µM by scratch assay. In HaCaT cells, 10 µM of SYN-1 peptide induced migration at 48 h in the absence of FBS (p = 0.02, Students'
Migration of HaCaT and SCC-9 cells treated with synthetic syndecan-1 peptide (SYN-1) and its scrambled peptide (control) in the concentrations of 1 µM, 10 µM and 100 µM were evaluated by scratch assay. SYN-1 did not induce migration in HaCaT cells at 24 h (A), but 10 µM of SYN-1 promoted migration at 48 h in absence of FBS (B), 1 µM of SYN-1 induced migration in HaCaT cells at 24 h (C) and 48 h (D), both in the presence of 1% FBS. SCC-9 cell migration was statistically significant at the concentration of 1 µM of SYN-1 in the absence of FBS at 24 h (E) and 48 h (F), but it was not statistically significant when compared to the scrambled peptide in the presence of 1% FBS at 24 h (G) and 48 h (H). Three independent experiments were performed with duplicates. Columns represent mean ± SD (n = 3) and * and ** indicate p<0.05 and p<0.01, respectively.
The effect of SYN-1 peptide on migration was also evaluated by transwell assay at concentration of 10 µM in absence of FBS. The results showed an increase of migration only in SCC-9 cells (p = 0.02, Students'
The migration of HaCaT and SCC-9 cells treated with synthetic syndecan-1-derived peptide (SYN-1) and its scrambled in a concentration of 10 µM in the absence of FBS was evaluated by transwell assay. The measurements were normalized with the control (scrambled peptide). SYN-1 did not induce migration in HaCaT cells (A). SCC-9 cell migration was statistically significant when compared to scrambled peptide (B). Three experiments were performed with triplicates. Columns represent mean ± SD (n = 3) and * indicate p<0.05.
One-way ANOVA analysis shows that PMA treatment did not change the mRNA expression levels of syndecan-1 in HaCaT and SCC-9 cells according to the time-course experiment (p>0.05). However, there were statistically significant differences in the expression levels of syndecan-1 between the cell lines at 0, 5 and 30 min upon PMA treatment (p = 0.009, 0.01 and 0.004, respectively) (
Ectodomain shedding of cell surface proteins, secreted proteins and proteolysis-derived fragments are soluble protein candidates to contribute to a diverse extracellular milieu and subsequently certain biological events related to cancer
Differences between these cell lines, with or without stimulation by PMA, were observed in migration, adhesion as well as in gelatinase activity (
Among the identified proteins, heparan sulphate proteoglycans, such as syndecan-1, were found only in the media of stimulated SCC-9 cells (
Interestingly, fragments of syndecan-1 were also found in higher abundance in stimulated SCC-9 cells (
To support a functional role for soluble syndecan-1 fragments, we demonstrated that the synthetic syndecan-1 peptide (SYN-1) was able to induce cell migration in both cell lines, as observed by a scratch assay (
The analysis of mRNA of syndecan-1 excluded the possibility that the increase of syndecan-1 in the media would be influenced by the increase of gene expression. We showed here that syndecan-1 mRNA expression levels did not change in a time-dependent manner (
In summary, this study demonstrated that the repertoire of secreted, shed and degraded proteins in the extracellular milieu of PMA-stimulated non-tumorigenic HaCaT and tumorigenic SCC-9 cells could be involved in fundamental cell processes and reveals that a fragment of syndecan-1 was able to induce cell migration.
The human OSCC cell line SCC-9 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured as recommended. SCC-9 cells originated from human squamous carcinoma from the tongue. The HaCaT cells, an immortalized but not transformed epithelial cell line
The HaCaT and SCC-9 cells were cultured until 80% of confluence in 500-cm2 plates, washed with serum-free media and then stimulated two times, with 12 h interval, with 50 ng/ml PMA (Sigma) diluted in DMSO. The same concentration of DMSO was used as control in the time 0 and after 12 h. After 24 h, the media were collected and the proteins and peptides were analyzed as described below.
The media were collected and the final concentration of 1 mM EDTA and 0.5 mM PMSF were added to the media. The protocol to obtain peptides and proteins was performed with few modifications
For protein analysis, an aliquot of 4.5 μl containing 15 μg of proteins of the resulting peptide mixture was evaluated as previously described
The spectra were acquired using software MassLynx v.4.1 and the raw data files were converted to a peak list format (mgf) without summing the scans by the software Mascot Distiller v.2.3.2.0, 2009 (Matrix Science Ldt.) allowing the label-free analysis, and searched against Human International Protein Database (IPI) v. 3.72 (86392 sequences, 35093930 residues; release date April, 2010) using Mascot engine v.2.3.01 (Matrix Science Ltd.), with carbamidomethylation as fixed modifications, oxidation of methionine as variable modification, one trypsin missed cleavage and a tolerance of 0.1 Da for both precursor and fragment ions. For the protein quantitation, the .dat files from Mascot output were analyzed in Scaffold Q+ (version 3_00_03, Proteome Software) and the quantitative value (normalized spectral counts) was obtained
The migration of HaCaT and SCC-9 cells was investigated through an in vitro monolayer assay. Cells grown in 12-well plates to confluence were scraped with a p200 pipette tip to create a cell-free area
The ability of HaCaT and SCC-9 cells to adhere to extracellular matrix proteins was evaluated in the adhesion assay
For 1-D zymography, the proteins (12 μg) in the conditioned media collected after cell treatments with 50 ng/ml PMA two times with 12 h interval were submitted to 1-D electrophoresis on 12% SDS-polyacrylamide gels containing 1 mg/ml gelatin under nonreducing conditions, and gelatinolytic activity was performed as previously described
For immunofluorescence assays, SCC-9 cells were cultivated in a CellCarrier (Perkin Elmer) 384 well plate. After PMA stimulation as described above, the cells were analyzed after 5 min, 30 min and 24 h. The cells were washed with PBS, fixed with 4% formaldehyde for 10 min, washed again and permeabilized with 0.5% Triton X-100 for 10 min. The cells were blocked with blocking solution (PBS containing 0.2% Triton and 3% non-fat dry milk) for 30 min and then incubated with the goat anti-syndecan-1 antibody (R&D Systems) diluted in blocking solution for 1 h. Cells were washed with PBS and incubated with an anti-goat Alexa Fluor 488-conjugated antibody (Invitrogen), diluted in blocking solution for 1 h. Finally, the cells were washed with PBS, incubated with DAPI (4′,6-diamidino-2-phenylindole) solution for 10 min, washed again and analyzed by the Operetta high content image system (Perkin Elmer). All pictures were acquired with the same contrast and brightness parameters. For automatic fluorescence quantification in the Operetta platform, DAPI staining was used for cell counting. Syndecan-1 Alexa Fluor 488 staining, restricted to the cell membranes, was defined by the most appropriate mask. Three independent experiments were performed.
The peptide of syndecan-1 identified by MS and manually validated (H-SQGLLDRKEVLGGVIAGGLVG-OH), named SYN-1, and the scrambled control (H-IGVGGLRELVKQLGDLGGVSA-OH) were chosen for functional experiments. The peptide was synthesized (Proteimax, São Paulo, Brazil) with the same sequence identified by mass spectrometry.
Migration of HaCaT and SCC-9 cells was performed as described before. Cells grown in 12-well plates to confluence were scraped with p200 pipette tip to create a cell-free area. The cells were washed three times with serum-free media to remove cell debris and incubated with serum-free media or 1% FBS containing the synthetic peptides SYN-1 and its scrambled in the concentrations of 1 µM, 10 µM and 100 µM. The migration was evaluated after 0, 24 and 48 h and analyzed by ImageJ software. Three independent experiments were performed with duplicates.
HaCaT and SCC-9 cells (7.5×104 cells) were plated in the top chambers of 8 μm pore transwells (HTS Transwell-96 Well Plate, Corning) in the serum-free culture medium after a starvation period of 18 h. The cells were allowed to migrate towards serum-free medium supplemented with 10 µM SYN-1 peptide or its scrambled over a period of 6 h. At the end of the assay, cells at the top chamber were removed with a cotton swab and the cells at the bottom of the filter were fixed with 10% formaldehyde for 10 min, washed with PBS and stained with 1% toluidine blue solution in 1% borax for 5 min. The dye was eluted using 1% SDS and the absorbance was measured at 620 nm. Three independent experiments were performed in triplicates.
In order to analyze the expression of syndecan-1, HaCaT and SCC-9 cells were cultured for 24 h in serum-free medium and treated with PMA during 5, 30 min and 24 h as described before. Total RNA was obtained using the TRIzol reagent (Invitrogen Corporation) and 2 µg of total RNA were used for retro-transcription using the First-Strand cDNA Synthesis Kit (GE Healthcare). Real-time quantitative PCR for syndecan-1 was performed using SYBR® Green PCR Master Mix (Applied Biosystems), and the dissociation curves were performed to confirm the specificity of products. The syndecan-1 forward primer was 5′- AGAAGAAGGACGAAGGCAGCTACT- 3′ and reverse primer was 5′- ATTCCTCCTGTTTGGTGGGCTTCT- 3′. The threshold cycles (CT) values of target gene were normalized relative to glyceraldehydes-3-phospate dehydrogenase gene, and relative expression ratios were calculated by the 2−ΔΔ Ct method. Three-independent experiments were performed with duplicates.
For the functional experiments, the assumptions of adherence of the errors to the Gaussian distribution were tested, for all response variables, using the Shapiro-Wilk Test. The GLIMMIX procedure of the SAS System (SAS Institute Inc. The SAS System, release 9.12. SAS Institute Inc., Cary:NC, 2008) were used to calculate the analysis of variance followed Student's
We thank Dr. Sandra Dias from Brazilian Biosciences National Laboratory for helping with immunofluorescence experiments. We thank Dr. Marcelo Corrêa Alves from “Luiz de Queiroz” College of Agriculture for helping with statistical analysis.