Inducibly-expressed Egln3 down-regulates Hif-2α, Oct4 and Klf5 in glioma cells.

<p>(A) Total cell lysates were prepared from Hu-glioma cells grown for 6hrs under hypoxia in the presence of 0–1µg/ml of Dox and analyzed for Egln3, Hif-1α and Hif-2α expression by Western. Changes in protein expression levels of Hif-1α ℓ (y-axis scale, 0–1.2), Hif-2 _ (y-axis scale, 0–1.2), Egln3 (y-axis scale, 0–60) were quantified relative to controls (hypoxia 6hrs, -Dox). Klf5 and Oct4 mRNA expression levels were determined by RT-QPCR (far right panel) performed on Hu-glioma cells Egln3 +/− 0.25µg/mL Dox that were cultured under hypoxia for 6hrs. (B) Rt-glioma cells Egln3 +/− 1µg/ml Dox were assayed for Egln3, Hif-1α and Hif-2α expression by Western. Klf5 and Oct4 mRNA expression analysis was conducted by RT-QPCR (far right panel). Hypoxic samples are shown; data are expressed as fold changes relative to control (hypoxia 6hrs, -Dox). Mean values +/− s.d. are shown; n = 3.</p

Hypoxic expression profile using an Human glioma cell model.

<p>Hu-glioma cells, Rt-glioma cells and Ms-NSCs were cultured under normoxic conditions and subjected to hypoxia for 6–48hrs, as indicated. (A) RT-QPCR was performed with primers specific for Egln1, Egln3, Klf5, Hif-1α, Hif-2α and Vegf. Data are expressed as fold changes relative to the normoxic condition for each respective cell type and normalized to β-actin mRNA. Mean values +/− s.d. are shown; n = 3. (B) Protein expression analysis of Hu-glioma cells and Ms-NSCs, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040053#pone-0040053-g001" target="_blank">Figure 1</a>.</p

Hif-2αinduces Klf5-expression by glioma cells.

<p>(A) Normoxic Rt-glioma cells +/− 1µg/mL Dox treatment for 0–18hrs were analyzed by Western for the expression of degradation-resistant Hif-1α^P564A and Hif-2α^P531A. (B, C) Normoxic Rt-glioma cells were induced with 1µg/ml Dox for 6hrs or 18hrs and analyzed for Vegf (B) or Klf5 (C) mRNA expression by RT-QPCR, respectively. Data are expressed as fold changes relative to no Dox controls. Mean values +/− s.d. are shown; n = 3. (D) Normoxic Rt-glioma cells were induced with 1µg/ml Dox for 18hrs and Klf5 protein expression assessed by Western.</p

Hydroxylase- dependent and independent roles of Egln3 in glioma progression.

<p>(A) Western analysis of Hu-glioma cells induced to express wild-type Egln3 or an hydroxylase defective mutant, Egln3^H196A with Dox (0.25µg/mL) for 6hrs under hypoxia <i>in vitro</i>. (B, C, D) Mock-treated Hu-glioma cells or Hu-glioma transfected with Dox-inducible Egln3 or Egln3^H196A constructs were injected subcutaneously into the flanks of NSG mice. Dox was administered once daily beginning 1 week following engraftment. Mean values +/– s.d. are shown; n = 3. (B) Quantification of mRNA expression in total tumor samples were determined by RT-QPCR. (C, D) Expression of Egln3 or the hydroxylase-defective Egln3^H196A decreased glioma progression. Tumor mass was determined 30 days post injection. The asterisks (**) or (***) indicates significance with a <i>p</i> value of ≤0.001 or ≤0.0001, respectively.</p

Hypoxic expression profile using a Rat glioma cell model.

<p>(A) Model for Egln3 function in glioma. (B, C) Rt-glioma cells or Rt-NSCs were subjected either to atmospheric O₂ levels, normoxia (N) or to 1% O₂, hypoxia (H) for the indicated time. mRNA (B) and protein (C) expression analysis of Egln1 Egln3c, Hif-1α, Hif-2α and Klf5 by RT-PCR and Western. β-actin served as a loading control.</p

Expression of Egln3 decreases tumor aggression, increases survival and normalizes glioma vascular architecture.

<p>(A) Timeline for the intracranial xenoengraftment of Hu-glioma initiating cells, Dox-induced expression of Egln3 in tumor cells and tumor analysis. (B) Kaplan-Meier survival curves for Hu-glioma (empty vector transfected; grey), Hu-glioma Egln3 + Dox (red), and Hu-glioma Hif-2α^P531A + Dox as a control (black); in total, n = 18). (C) Fluorescent images of intra-tumor blood capillaries. Gliomas originating from Hu-glioma initiating cells (empty vector transfected) and Hu-glioma Egln3 + Dox cells were allowed to develop until the onset of neurological symptoms and labeled with anti-CD31 (PECAM) antibodies and DAPI. Scale Bar  = 100µm. (D) Percentage of CD31-labeled blood capillaries that exhibited diameters of 1–25µm, 26–50µm or 51–90µm relative to internal scale bars in non-cancerous adult mouse brain, Hu-gliomas (empty vector transfected) and Hu-gliomas Egln3 + Dox after tumor progression elicited neurological symptoms. >500 capillaries of 10 randomly chosen tumor sections were chosen from 3 glioma engrafted mice for each experimental group. Mean values +/− s.d. are shown; n = 4. (E) Rt-glioma (empty vector transfected) and Rt-glioma Egln3 + Dox were allowed to develop until the onset of neurological symptoms and fluorescently labeled with the endothelial-specific probe, isolectin GS-IB₄ (Lectin) and DAPI. Scale Bar  = 100µm. (F) Percentage of Lectin-labeled tumor blood capillaries that exhibited diameters of 1–25µm, 26–50µm or 51–90µm in empty vector transfected Rt-gliomas and Rt-gliomas Egln3 +/− Dox after tumor progression elicited neurological symptoms. Mean values +/− s.d. are shown; n = 4. (G) Empty vector transfected Rt-glioma and Rt-glioma Egln3 +/− Dox tumors were allowed to progress until neurological symptoms were noted. Quantification of Vegf, Oct4 and Klf5 mRNA expression in total tumor were determined by RT-QPCR. Data are expressed as fold changes relative to empty vector transfected Rt-glioma tumor samples. Mean values +/− s.d. are shown.</p

Suppression of Hif-2α reduces Klf5 expression in Hu-glioma cells.

<p>Hu-glioma cells expressing luciferase shRNA (control) or Hif-2α shRNA (shHif-2α) were subjected to hypoxia for 6hrs and analyzed for gene expression by RT-QPCR (A) and Western (B). Data are expressed as fold changes relative to shRNA controls.</p