Supplementary Tables
Supplementary Table S1. List of primers used in the present study.
Supplementary Table S2. Correlation between Brachyury expression and molecular markers.
Supplementary Figure Legends
Supplementary Figure S1. Brachyury protein staining analysis in PCa tissues. A) Whisker-box plot shows the distribution of semi-quantitative scores evaluated in 13 normal tissues and in 211 non-neoplastic adjacent tissue microarray samples. Brachyury staining is similar in normal gland and adjacent tissues both in intensity and extension. Figure linked to Figure 1. B) Left upper corner: representative image of a negative normal adjacent tissue. In the right upper corner figure we can obverse and increase in intensity from non-tumoral (PIN lesion) to tumoral (PCa) lesions in the same case. We found a high stromal expression in non-neoplastic and PIN tissues at variance with absence in PCa tissues. Brachyury is expressed in stromal cells in non-neoplastic tissues and PIN lesions and its expression decreases in PCa tissues (left and right bottom figures, respectively). Magnification of 200X. Figure linked to Figure 1 and 2.
Supplementary Figure S2. The percentage of Brachyury-positive cases increases proportionally with Gleason score aggressiveness. Protein Brachyury-positive tumors significantly increased with Gleason score. Microarray gene expression analysis for Brachyury levels in five different data sets shows the same significance as in the tissue microarray cohort analyzed in present study. The. *, p<0.05; **, p<0.01; ***, p<0.001. Figure linked to Table 1 and 2.
Supplementary Figure S3. Brachyury characterization and modulation in prostate cancer cell lines. A) Prostate cell line characterization for Brachyury expression using conventional RT-PCR, western blot and immunofluorescence assays. PNT2 and 22RV1 are Brachyury-negative. DU145 cell line has residual nuclear Brachyury expression. PC3 and LNCaP are strongly positive for Brachyury. B) Brachyury expression modulation was evaluated by RT-PCR and immunofluorescence. Brachyury was successfully overexpressed in 22RV1 and DU145 and depleted in PC3 cancer cell lines. In 22RV1 pcBrachyury cells and in PC3 sh.Brachy.1 cells it was possible to detect some non-transfected cells confirming the specificity of the anti-Brachyury antibody.
Supplementary Figure S4. Brachyury overexpression in DU145 metastatic cell line increases prostate cancer cell aggressiveness. We validate the in vitro findings for primary PCa cell line (22RV1) and for bone metastatic cell line (PC3) in a third PCa cell line (brain metastatic DU145 cell line). Overexpression in DU145 cells (pcBrachyury; dotted red lines and black bars) promotes and increased cell viability, proliferation, migration and invasion capability compared with control cells (T/04; full blue line and with bars). *, p<0.05; **, p<0.01; ***, p0.001. Figure linked to Figure 3.
Supplementary Figure S5. Brachyury overexpression modulates expression of EMT and stem cell markers in prostate cancer cell lines. A) Influence of Brachyury modulation in epithelial (E-cadherin), mesenchymal (Snail, N-cadherin, vimentin, fibronectin), migration/invasion (MMP14) and stem cell marker (CD44) in prostate cell lines with differential Brachyury expression. B) Densimetric semi-quantification of bands (A) using Image J software. Data represents at least 2 independent replicates.