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

Quantitative PCR results of <i>NANOG1</i> and <i>NONOGP8</i>.

<p>Compared to the cells under normoxia, there are elevated <i>NANOG1</i> and <i>NONOGP8</i> expressions in the cells under 7% O₂ for both cell lines, with up to 1.8-fold increase <i>NANOG1</i> expression and up to 2.5-fold <i>NONOGP8</i> increase in both cell lines; the cells under 1% O₂ express even higher levels of <i>NANOG1</i> and <i>NONOGP8</i>, with 2.6-fold and 3.1-fold increase in <i>NANOG1</i> expression in the PC-3 and DU145 cell lines, respectively, and with 6-fold and 10-fold increase in <i>NONOGP8</i> expressions in the PC-3 and DU145 cell lines, respectively.</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

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

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 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>(A–C) CD44, CD24 and CD90 expressions were analyzed by flow cytometry in LNCaP and PC-3 cells cultivated with/without DHT in 10 nM for 48 hours. CD44 was induced by DHT treatment in both cell lines (A). No statistically significant difference of CD24 expression level was shown in both cell lines (B). CD90 expression was significantly increased by DHT stimulation in both cell lines (C). (* means <i>P</i><0.05).</p

Clinical and pathologic characteristics for 117 patients with malignant prostate cancer.

<p>PSA, prostatic specific antigen.</p

DHT induces cell growth and clonogenicity in prostate cancer cell lines.

<p>(A) Cell growth curves show statistically significant difference in LNCaP cells with/without DHT treatment, but not in PC-3 cells (* means <i>P</i><0.05). (B) Representative photographs of colony formation in both cell lines demonstrate that more colonies were formed by 1 nM DHT treatment and even more colonies were obtained by 10 nM DHT treatment (bar scale: 50 mm). (C) Histograms of colony formation efficiency show statistically higher efficiencies in the cells treated with low concentration of DHT (<i>P</i><0.01), and even higher efficiencies in the cells by high concentration of DHT (<i>P</i><0.001). (D) Representative photographs for sphere formation for both cell lines in the cells with/without DHT treatment (bar scale: 50 µm). (E) Histograms for sphere formation efficiency show higher efficiencies in the cells added with 1 nM DHT (<i>P</i><0.05), and even higher efficiencies in the cells stimulated with 10 nM DHT (<i>P</i><0.01).</p