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1.
Figure 7

Figure 7. From: Stanniocalcin 1 and Ovarian Tumorigenesis.

Model of stanniocalcin 1 (STC1)–mediated ovarian tumorigenesis. RAS or possibly other oncogenic events (eg, mutation in p53) activate the expression of STC1 protein and increase its secretion. The increased level of secreted STC1 protein inhibits apoptosis by an as yet unknown mechanism to increase the expression of the antiapoptotic proteins Bcl-2 or Bcl-XL and to promote cell cycle progression by increasing the expression level of cell cycle regulatory protein cyclin B1, cyclin A, cyclin E, and cyclin-dependent kinase 2 (CDK2).

Guangzhi Liu, et al. J Natl Cancer Inst. 2010 June 2;102(11):812-827.
2.
Figure 5

Figure 5. From: Stanniocalcin 1 and Ovarian Tumorigenesis.

Apoptosis of ovarian cancer cells and stanniocalcin 1 (STC1) expression. A) Percentage of apoptotic cells. Apoptosis was measured by flow cytometry in cells stained with annexin V. B and C) Apoptosis in ovarian xenograft tumors from nude mice subcutaneously injected with T29H-STC1i or control T29H-scr cells or HEY-STC1i or control HEY-scr cells. Apoptosis was assessed with a bromodeoxyuridine-coupled terminal deoxynucleotidyltransferase deoxyuridine triphosphate nick end labeling assay. B) Quantitative analysis of apoptotic cells. Error bars = 95% confidence intervals (CIs). C) Representative fluorescence micrographs. Propidium iodide (PI)-–labeled nuclei = red; apoptotic cells labeled with bromolated deoxyuridine triphosphate (Br-dUTP) = green. Micrographs were taken at ×400 magnification. Scale bars = 50 μm. D and E) Percentage of apoptotic cells in cell culture after treatment with neutralizing anti-STC1 antibody. D) T29H cells. E) HEY cells. Cells were treated with anti-STC1 antibody (STC1Ab) or a control IgG, as indicated. One pair of samples was also treated with STC1 protein (20 nM/mL) for 24 hours after addition of STC1 antibody (labeled 24h + STC1 protein). Error bars = 95% CI.

Guangzhi Liu, et al. J Natl Cancer Inst. 2010 June 2;102(11):812-827.
3.
Figure 4

Figure 4. From: Stanniocalcin 1 and Ovarian Tumorigenesis.

Cell cycle distribution of ovarian cancer cells and stanniocalcin 1 (STC1) expression. Cells with overexpression of STC1 (T29-STC1, T80-STC1, and OVCA420-STC1) and their corresponding controls (T29-vector, T80-vector, and OVCA420-vector) or with silenced expression of STC1 (T29H-STC1i, HEY-STC1i, and SKOV3-STC1i) and their corresponding controls (T29H-scrambled small interfering RNA [scr], HEY-scr, and SKOV3-scr) were tested. A) Cell cycle distribution of cells that overexpress STC1 or cells in which STC1 expression was silenced and their control cells. The cell cycle distribution of cells stained with propidium iodide was determined by flow cytometry. B) Quantitative analysis of cell cycle distribution. Data from three independent experiments were used in this analysis. Error bars = 95% confidence intervals. C and D) Immunoblot analysis of cell cycle regulatory proteins (cyclin E, cyclin-dependent kinase 2 [CDK2], cyclin A, cyclin B1, cyclin D1, and CDK4). Expression of these proteins was assessed by immunoblot analysis with the corresponding antibodies. Bands were visualized by electrochemiluminescence chemiluminescence. β-Actin was used as the loading control. C) Cells with STC1 overexpression (STC1+) or their control cells (STC1−). For each protein, the ratio of expression in each pair of cells (ie, STC1 cDNA−/STC1 cDNA+) is shown under the corresponding immunoblot bands. D) Cells whose STC1 expression was silenced (STC1i+) or their corresponding control cells (SCT1i−). For each protein, the ratio of expression in each pair of cells (ie, STC1i−/STC1i +) is shown under the corresponding immunoblot bands. F = full length form of cyclin E; L or * = low molecular weight of cyclin E; PI = propidium iodide.

Guangzhi Liu, et al. J Natl Cancer Inst. 2010 June 2;102(11):812-827.
4.
Figure 6

Figure 6. From: Stanniocalcin 1 and Ovarian Tumorigenesis.

Stanniocalcin 1 (STC1) expression, carboplatin-mediated resistance, and apoptotic signaling pathway. Cell lines with ectopic expression of STC1 (T29-STC1 and T80-STC1) and their corresponding controls (T29-vector and T80-vector) or cells whose STC1 expression was silenced with STC1 small interfering RNA (siRNA) (T29H-STC1i and HEY-STC1i) and their corresponding controls (T29H-scr and HEY-scr) that contained scrambled siRNA were used in these assays. A and B) Chemosensitivity to carboplatin treatment. Cells (as indicated) were treated with carboplatin as indicated concentration for 12 hours, the drug was washed out, and cell viability was measured by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 72 hours later. Data are optical density (OD) absorbance at 570 nm, which have been subtracted from the background OD at 490 nm. Error bars = 95% confidence intervals (CIs). C) Half-maximal inhibitory concentration (IC50) values of carboplatin. IC50 values were determined by use of the MTT, as described above. Error bars = 95% CI. D) Expression of proteins involved in regulation of the apoptotic pathway, including BCL-2, BCL-XL, BAD, pBAD, BAX, and caspases-1, -3, -7, -8, -9, and -10 in cells with altered STC1 protein expression. Regulatory proteins were detected by immunoblot analysis with the corresponding antibodies and visualized by electrochemiluminescence chemiluminescence. β-Actin was the loading control. For each protein, the ratio of expression in each pair of cells (ie, SCT1+/SCT1− or STC1i−/STC1i +) is shown under the corresponding immunoblot bands. * = cleaved caspase-8; ** = cleaved caspase-9; *** = cleaved caspase-3; arrow = procaspase or cleaved caspase.

Guangzhi Liu, et al. J Natl Cancer Inst. 2010 June 2;102(11):812-827.
5.
Figure 2

Figure 2. From: Stanniocalcin 1 and Ovarian Tumorigenesis.

Stanniocalcin 1 (STC1) expression and proliferation of ovarian cancer cells. STC1-overexpressed cells (T29-STC1, T80-STC1, and OVCA420-STC1) and their corresponding control cells (T29-vector, T80-vector, and OVCA420-vector), STC1-silenced cells (T29H-STC1i, HEY-STC1i, and SKOV3-STC1i) and their corresponding control cells that contained scrambled small interfering RNA (T29H-scr, HEY-scr, and SKOV3-scr), and HRAS-silenced cells (SKOV3-HRASi) and their control cells (SKOV3-scr) were used. A) Immunoblot analysis of STC1 protein expression in STC1-overexpressed cells (T29-STC1, T80-STC1, and OVCA420-STC1) and their corresponding control cells (T29-vector, T80-vector, and OVCA420-vector), STC1-silenced cells (T29H-STC1i, HEY-STC1i, and SKOV3-STC1i) and their corresponding control cells (T29H-scr, HEY-scr, and SKOV3-scr), and HRAS-silenced cells (SKOV3-HRASi) and their control cells (SKOV3-scr), and ovarian cancer cells. Whole-cell lysates were analyzed by goat anti-STC1 antibody and visualized by electrochemiluminescence (Amersham Biosciences). B) Cell proliferation and the level of STC1 protein expression. Cell proliferation in STC1-overexpressed cells and STC1-silenced cells and their corresponding control cells was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at an optical density (OD) absorbance of 570 nm by using a Flexmap 3D High-Throughput Multiplexing System. Open bars = control cells; filled bars = cells with overexpressed or silenced expression of STC1; error bars = 95% confidence intervals (CIs). Data were from three independent experiments with triplicate samples. C) Cell migration and the level of STC1 protein expression. The migration assay was performed in 24-well two-chamber plates with an high throughput screening multi-well insert in medium containing fibronectin. The number of cells that migrated through the membrane was counted after staining with a Giemsa solution. Open bars = number of migrated cells in control cell lines; filled bars = number of migrated cells in cell lines with overexpressed or silenced expression of STC1; error bars = 95% CIs. Data from three independent experiments are shown. D) Anchorage-independent colony formation on soft agar in association with STC1 protein expression. The anchorage-independent assay was performed with 5 × 104 cells per 60-mm dish in medium containing 0.35% agarose. All colonies with more than 100 cells were counted. Error bars = 95% CIs. Data from three independent experiments are shown. SC = subcutaneous.

Guangzhi Liu, et al. J Natl Cancer Inst. 2010 June 2;102(11):812-827.
6.
Figure 1

Figure 1. From: Stanniocalcin 1 and Ovarian Tumorigenesis.

Stanniocalcin 1 (STC1) protein expression in immortalized ovarian surface epithelial cells (T80 and T29 cells), RAS-transformed ovarian surface epithelial cells (T80H, T80K, T29H, and T29K cells), and ovarian cancer cell lines (SKOV3, HEY, OVCA420, OVCA432, and OVCA433 cells), ovarian tissues (normal ovary, benign cyst, borderline tumor, endometroid carcinoma, and high-grade serous carcinoma), and serum from ovarian cancer patients and control women. A) Immunoblot analysis of whole-cell lysates from ovarian cancer cells for STC1 protein expression. Each lane contained 50 μg of cell lysate protein. B) Immunoblot analysis of STC1 protein in the conditioned culture medium from ovarian cancer cell lines. Each lane contained 30 μL of conditioned medium. In both (A) and (B), immunoblots were probed with goat anti-STC1 antibody and the secondary antibody was donkey anti-goat IgG conjugated with horseradish peroxidase. Results were visualized by electrochemiluminescence (Amersham Biosciences). C) Enzyme-linked immunosorbent assay (ELISA) detection of STC1 protein in conditioned culture medium from ovarian cancer cell lines. Data are the mean of three independent experiments with triplicate samples. Error bars = 95% confidence intervals (CIs). D) ELISA detection of STC1 protein in serum from 53 patients with ovarian cancer and 73 healthy women (control subjects). Data are the concentration of STC1 protein. The mean STC1 level and its 95% CIs are shown at the bottom. The P value for the comparison of serum levels STC1 in patients with ovarian cancer with that in control subjects was .021 (χ2 test). All statistical tests were two-sided. E) Immunohistochemical staining of ovarian tissues for STC1 protein expression. Tissues were stained with goat anti-STC1 antibodies and visualized with donkey anti-goat secondary antibody. Arrows indicate cytoplasmic expression of STC1 in high-grade serous carcinoma. Scale bars = 50 μm.

Guangzhi Liu, et al. J Natl Cancer Inst. 2010 June 2;102(11):812-827.
7.
Figure 3

Figure 3. From: Stanniocalcin 1 and Ovarian Tumorigenesis.

Xenograft ovarian tumor burden in mice and stanniocalcin 1 (STC1) expression. Cells with overexpression of STC1 (T29-STC1, T80-STC1, and OVCA420-STC1) and with silenced expression of STC1 (T29H-STC1i, HEY-STC1i, and SKOV3-STC1i) and their corresponding control cells were used to establish tumors. A and B) Volume of tumors produced by subcutaneous injection for T29-STC1 cells or T29-vector control cells (A) and for T80-STC1 cells or T80-vector control cells (B). Each cell line was injected into the bilateral flanks of eight mice, which produced 16 subcutaneous tumors (two tumors per mouse). Error bars = 95% confidence intervals (CIs). C) Hematoxylin–eosin staining or immunohistochemical staining for STC1 protein, SV40 large T antigen, p53, and CA 125 in xenograft tumors produced by intraperitoneal injection of T29-SCT1 or T80-STC1 cells. Antibodies against STC1, SV40 large T antigen, p53, or CA 125 were used. Antibody binding was detected by using avidin–biotin–peroxidase and visualized with the chromogen 3,3′-diaminobenzidine. Scale bars = 50 μm. D) Immunofluorescence staining for CA 125 in T29-STC1 and T29-vector control cells and in T80-STC1 and T80-vector control cells. Anti-CA 125 monoclonal antibody was used, and the secondary antibody was donkey anti-mouse IgG conjugated with fluorescein isothiocyanate (green). β-Actin was detected with a polyclonal primary antibody and donkey anti-goat secondary antibody conjugated with Texas red to show the actin cytoskeleton. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Overlaid images were merged to show CA 125 (green), cytoskeletal marker actin (red), and nuclei (blue). Scale bars = 50 μm. E) Growth of tumors produced by subcutaneous injection of mice with OVCA420-STC1 cells or vector control cells. A total of six mice were used for each cell line, with each mouse receiving injections at two sites to form two tumors per mouse. Error bars = 95% CI. F–H) Growth of tumors produced by subcutaneous injection of mice with cells transfected with small interfering RNA (siRNA) against STC1 (eg, T29H-STC1i) or corresponding control scrambled siRNA (eg, T29H-scr). A total of six mice for T29H-STC1i, HEY-STC1i, T29H-scr, and HEY-scr cells, or eight mice for SKOV3-STC1i and SKOV3-scr cells were used and with each mouse receiving injections as two sites to form two tumors per mouse. Error bars = 95% CI. F) T29H-STC1i cells and T29H-scr control cells. G) HEY-STC1i cells and HEY-scr control cells. H) SKOV3-STC1i cells and SKOV3-scr control cells. I) STC1 expression in xenograft tumors generated by the following cells: HEY-STC1i and control HEY-scr cells and by SKOV3-STC1i and control SKOV3-scr cells. IHC = immunohistochemistry.

Guangzhi Liu, et al. J Natl Cancer Inst. 2010 June 2;102(11):812-827.

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