Comparison of clinical and pathologic characteristics by tumor SHBG intensity.

<p>Muci, Mucinous tumor; Endo, Endometrioid; Clea, Clear cell; Mixe, Mixed epithelial tumor; Undi, Undifferenciated tumor.</p

OS and PFS for the ovarian carcinoma patients in 3 SHBG IHC groups.

<p>For both OS (A) and PFS (B) curves, weakly (IHC score 1), moderately (IHC score 2) and strongly (IHC score 3) SHBG expressing groups were marked by blue, green and brown lines respectively. No statistically significant difference was obtained both for OS (<i>p</i> = 0.220) and PFS (<i>p</i> = 0.132).</p

IHC SHBG staining in clinical ovarian carcinoma samples.

<p>Representative weak (A), moderate (B, moderately differentiated tumor) and strong (C, poorly differentiated tumor) are shown. Stronger SHBG immunostaining is shown at the invasive front of a poor-differentiated ovarian carcinoma (D). Negative control performed in human liver tissue with the same concentration of non-immune goat IgG shows no staining in the liver cells (E). Positive control performed with liver tissue shows the positive cytoplasm SHBG staining. Endothelial cells are also positive for SHBG. All photos were taken at 200×.</p

Hypoxic effects on cell proliferation, colony formation and cell cycle.

<p>(A) Cell proliferation curves show no statistical difference for cell growth under different oxygen tensions (<i>P>0.05</i>). (B) Colony formation assay for both cell lines shows more colonies in the cells pre-treated at 7% O₂ for 48 hours and even more colonies in the cells pre-treated under 1% O₂ (C) Histograms for the colony formation efficiency shows statistically higher efficiency in the cells pre-treated under hypoxia (7% or 1% O₂) for both cell lines (<i>P</i><0.0001). (D) Flow cytometry shows extended G₀/G₁ stage for both cell lines which have been cultivated under 1% O₂ for 48 hours, in comparison to the cells always kept under normoxia. (E) Statistical analyses reveal significantly extended G₀/G₁ stage in the cells cultivated under hypoxia for both cell lines (<i>P</i><0.05).</p

CD44^bright cells show stem-like properties.

<p>(A) For both PC-3 and DU145 cell lines, more colonies are shown in the CD44^bright cells than the CD44^dim cells under nomoxia condition, while even more colonies can be seen in the CD44^bright cells pretreated under 1% O₂ for 48 hours. (B) Histograms for the colony formation efficiency show statistically significantly higher efficiency in the CD44^bright cells than the corresponding CD44^dim cells (<i>P</i><0.0001 for both cell lines), and even higher level efficiency in the hypoxia- pretreated CD44^bright cells in comparisons with the CD44^bright cells without hypoxic pretreatment (<i>P</i><0.05 for both cell lines). (C) Sphere formation assay shows spheres in the CD44^bright cells in both normoxia and hypoxic pretreated cells while there is no qualified sphere in the corresponding CD44^dim cells for both cell lines. (D) Histograms show sphere formation efficiency in the CD44^bright and CD44^dim cells with and without hypoxia pretreatment.</p

(A) Known CD117-positive seminoma tissue is always positive for CD117 and used as positive control in this study.

<p>(B) Section from the same seminoma tissue is negative by incubating with the corresponding non-immune rabbit IgG in the same concentration, instead of rabbit anti-CD117 antibody. Both pictures were taken at 200×.</p

CD44^bright cells are mainly positive for ABCG2, Oct3/4 and Nanog.

<p>(A) Double staining of CD44 and ABCG2 surface markers with flow cytometry assay shows higher levels expressions of these factors in both cell lines under hypoxia for 48 hours. (B) The CD44^bright cells under normoxia express higher levels of ABCG2, Oct3/4 and Nanog, but the CD44^dim cells under the same normoxia condition express very low levels of these factors. The CD44^bright cells pretreated under 1% O₂ for 48 hours show even higher levels of these factors compared to the CD44^bright cells cultivated under normoxia.</p

Stem-like phenotype analyses by flow cytometry.

<p>PC-3 and DU145 cells were cultivated in 1% O₂ and 20% O₂ coditions for 48 hours to analyze stem-like phenotype through flow cytometry assay. (A) The representative images show that side population cells were induced in both cell lines after hypoxic treatment. (B) Statistic analyses show significant difference in side population. (C) Flow cytometry analyses show higher levels of ABCG2 expression intensity in both cell lines under hypoxia. (D) Histogram shows statistically significant difference in ABCG2 expression. (E) Flow cytometry analyses show higher CD44 expression intensity in both cell lines under hypoxia. (F) Histogram shows statistically significant difference in CD44 expression.</p

Hypoxia increases the expressions of Oct3/4 and Nanog.

<p>(A) In comparison to the cells cultivated at 20% O₂, the cells cultivated at 7% O₂ show higher levels of Oct3/4 and Nanog expressions, and the cells cultivated at 1% O₂ show the highest levels of Oct3/4 and Nanog expressions in PC-3 and DU145 cell lines by both RT-PCR and immunoblotting. (B) Immunocytochemistry reveals corresponding higher levels of Oct3/4 and Nanog expressions at 7% O₂ and the highest levels of Oct3/4 and Nanog expressions at 1% O₂, in comparison to the cells cultivated at 20% O₂ for both cell lines. Human seminoma tissue sections were used as positive controls for these two antibodies. All photos were originally taken at 200× and all the insets were taken at 400×.</p

Hypoxia increases the expressions of HIF-1α and HIF-2α.

<p>(A) In comparison to the cells cultivated at 20% O₂, the cells cultivated at 7% O₂ show higher levels of HIF-1α and HIF-2α, and the cells cultivated at 1% O₂ show the highest levels of HIF-1α and HIF-2α by both RT-PCR and immunoblotting. (B) Immunocytochemistry reveals higher levels of HIF-1α and HIF-2α expressions in the cells cultivated under 7% O₂ and the highest levels of HIF-1α and HIF-2α expressions in the cells cultivated under 1% O₂ for both cell lines. The breast carcinoma sections were used as positive controls for both HIF-1α and HIF-2α. All photos were originally taken at 200× and all the insets were taken at 400×.</p