Figure 1.
Expression of SATB1 in various representative benign, low-grade and high-grade human prostate tumor specimens.
(A) Protein expression of SATB1 in various grades of prostate tumors and benign tissue was analyzed by Western blotting; cytokeratin18 expression served as loading control. (B) SATB1 mRNA expression in various grades of prostate tumors and benign tissue; GAPDH mRNA expression served as loading control. (C) SATB1 protein expression in paired benign and cancer specimens from same individual was analyzed for cytoplasmic and nuclear distribution. Densitometric analysis for each blot is shown in the right panel. Details are described in ‘materials and methods’ section.
Figure 2.
Expression of SATB1 in various representative human prostate specimens.
(A) Paraffin-embedded (4.0 µm) sections from benign and prostate cancer of various Gleason scores were used for SATB1 expression by immunohistochemistry. A strong nuclear and cytoplasmic staining was observed in Gleason score 3+3, whereas increased nuclear SATB1 staining was observed in high-grade cancer (Gleason score 3+4 and 4+5, respectively). Magnified at x20 and x40 (B) Statistical analysis of SATB1 nuclear presence was performed by comparing SATB1 positive nuclear stained cells from various locations from benign, score 5–6, score 7–8 and score 9–10 specimens. Data represents the mean±SE. *P<0.05 versus corresponding control. P<0.05, Details are described in ‘materials and methods’ section.
Figure 3.
SATB1 protein expressions in virally transformed prostate epithelial cells, and various prostate cancer cells.
(A) Western blotting for SATB1 protein expression in various prostate cancer cells; CA-HPV-10, DUPro, DU145, PC-3, PC-3M, LNCaP and C4-2B including transformed epithelial (PZ-HPV-7), cells. (B) Cytoplasmic and nuclear SATB1 protein expression was analyzed in prostate cancer parental cell lineage and their aggressive subclones LNCaP and C4-2B; PC-3 and PC-3M which represented higher nuclear presence of SATB1 in aggressive subclones. Histone H4 served as loading control. Densitometric analysis for each blot is shown in the right panel. Details are described in ‘materials and methods’ section.
Figure 4.
Effect of suppression of SATB1 expression by small hairpin RNA (shRNA) in prostate cancer PC-3M cells.
Prostate cancer PC-3M cells were transfected with and without DNA (mock), or the SATB1-targeted shRNA vector 1and 2 and continued in culture for 24 h. (A) SATB1, E-cadherin, MMP9 and <beta>-actin total protein expressions were analyzed by Western blotting. (B) SATB1 silencing in PC-3M cells was associated with low invasive and migration potential. Bars represents the mean±SE of three different assays. **P<0.001 versus control. (C) Representative PC-3M cells images are shown with and without SATB1 shRNA transfection. Details are described in ‘materials and methods’ section.
Figure 5.
Overexpression of SATB1 in transformed normal prostate epithelial PZ-HPV-7 cells.
PZ-HPV-7 cells were transfected with and without DNA (mock), or the SATB1-targeted vector 1 and continued in culture for 24 h. (A) SATB1, E-cadherin, MMP9 and Histone H4 protein expressions were determined in the cytosolic and nuclear fraction by Western blotting. SATB1 overexpression resulted in decrease E-cadherin expression and upregulated MMP9 expression. (B) Overexpression of SATB1 in PZ-HPV-7 cells was associated with increased invasive potential. Bars represents the mean±SE of three different assays. **P<0.001 versus control. (C) Representative PZ-HPV-7 images with and without SATB1 overexpression vector trasfection. Details are described in ‘materials and methods’ section.
Figure 6.
Effect of SATB1 knock down in tumor growth in athymic nude xenograft.
(A) SATB1 knockout in PC-3M cells resulted in decrease in tumor volume. (B) Photograph of tumor xenograft in PC-3M and SATB1-KO tumors. (C) Tumors were excised, weighed, and measured at the termination of the study. Tumor weight was significantly reduced in SATB1-KO tumors in athymic nude mice. Approximately 1×106 cells were injected in the flanks of each mouse to initiate ectopic prostate tumor as described in ‘materials and methods’. Tumor size were measured twice weekly in two dimensions throughout the study. Tumor volume (mm3) and wet weight of tumors are represented as the mean of 8–10 tumors in control PC-3M and SATB1-KO PC-3M tumors. **P<0.003; Bars; Mean ± SE. (D) Western Blots for PCNA, E-cadherin, MMP9, and SATB1 in tumor lysates from PC-3M and SATB1-KO tumors. The blots were stripped and reprobed with anti-<beta>-actin antibody to ensure equal protein loading by a representative blot. Details are described in ‘materials and methods’ section.