Role of EBS4 in the activation of PTPRZ1 promoter by HIF-2α.

<p>(A) Effects of EBS4 or EBS5 deletion on the response of the PTPRZ1 promoter to HIF-2α. HEK293T cells were co-transfected with 300 ng of PTPRZ1-250WT, EBS4D, or EBS5D promoters and 50 ng of a internal β-gal control plasmid in the presence of 250 ng of an expression plasmid encoding HIF-1α, HIF-2α or pcDNA3.1 empty control vector. Results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009641#pone-0009641-g002" target="_blank">Fig 2</a>. (B). Binding of HIF-2α and HIF-1α to the PTPRZ1 promoter in the region near EBS4, EBS5, HRE4, and HRE5 <i>in vivo</i>. The chromatin immunoprecipitation assay was performed with HEK293T cells transfected with HIF-1α or HIF-2α respectively. Pre-cleared chromatin was immunoprecipitated with anti-HIF-2α or anti-HIF-1α antibody or normal rabbit IgG. After reversal of cross-linking, the DNA was analyzed by PCR. The primer set for PCR were designed to cover the EBS4, EBS5, HRE4, and HRE5 sites (C) Binding of ELK1 to the PTPRZ1 promoter. Experiment performed as in 7B except that the HEK293T cells were transfected with an Elk-1 expression vector and anti-ELK1 antibody or normal rabbit IgG was utilized.</p

HIF-1 and HIF-2 bind to PTPRZ1 oligonucleotide probes containing HRE4.

<p>DIG-labeled synthetic oligonucleotide containing HRE4 was incubated with nuclear extracts (NE) from normoxic (N), 16 hour hypoxic (H) or HIF-transfected (HIF-1α or HIF-2α) HEK293T cells and analyzed on a non-denaturing polyacrylamide gel. Where indicated, unlabeled wild type (WT) or mutant (Mut) oligonucleotides (at 50x and 100x of the labeled probe) were added to the binding reaction. Protein-DNA complexes were separated, blotted to a nylon membrane, and probed with anti-digoxigenin antibody conjugated to alkaline phosphatase. Comp denotes unlabeled probe used for binding competition. The sequence of the probe and WT and Mut competing oligonucleotides used is shown at the bottom. WT HRE4 sequence is underlined, and nucleotide changes in the Mut sequence are shown in bold. The positions of the DIG-labeled HIF complexes and free probe are indicated with arrows.</p

siRNA to ELK1 inhibits PRPRZ1 activation.

<p>(A) Immunoblotting analysis for ELK1, β-Actin, and GAPDH in HEK293T cells transfected with ELK1 scrambled siRNA (60 nM), β-Actin siRNA (60 nM), or ELK1 siRNA (60 nM). (B) ELK1 mRNA as determined by quantitative RT-PCR from HEK293T cells 48 hours after transfection with ELK1 scrambled siRNA, β-Actin siRNA, or ELK1 siRNA. (C) β-Actin mRNA under similar conditions. (D) PTPRZ1-250 promoter activity in HEK293T cells following transfection with HIF-2α plasmid alone or HIF-2α plasmid and either scrambled siRNA, or ELK1 siRNA. Data is presented as fold induction over vector control after normalization to β-gal. Bars represent mean and standard deviation of 3 determinations.</p

Comparison of the activation of truncated forms of PTPRZ1 luciferase reporter by HIF-1α and HIF-2α and their degradation-resistant forms.

<p>(A) Hep3B or (B) HEK293T cells were co-transfected with 300 ng of each PTPRZ1 promoter and 50 ng of an internal β-gal control plasmid in the presence of 250 ng of an expression plasmid encoding HIF-1α, drHIF-1α, HIF-2α, drHIF-2α, or pcDNA3.1control. Results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009641#pone-0009641-g002" target="_blank">Figure 2</a>.</p

PTPRZ1 luciferase promoter constructs showing the location of the potential hypoxia response elements (HRE) and Ets binding sequences (EBS).

<p>Each HRE is denoted as a square and each EBS as a plus sign. The TATA box and the ATG start site are indicated in the PTPRZ1-250 promoter. Each promoter construct extends 57 bp into the PTPRZ1 coding region prior to the luciferase (LUC) sequence except for the PTPRZ1-250 promoter, which stops at the ATG. The HRE consensus sequences and direction of each HRE are also indicated. The core HRE sequences are: HRE1, CCGTG; HRE2, CACGC; HRE3, CACGC; HRE4, CACGCACG; HRE5, CACGG.</p

Generation of BCBL-1 and BC-3 cells with stable HIF-1α knockdown.

<p><b>(A)</b> Protein levels of HIF-1α in nuclear extracts of BJAB, BCBL-1, and BC-3 cells measured by Western blot analysis after 24 hours in normoxia. Tata-binding protein (TBP) is used as a loading control. BCBL-1 and BC-3 cells were transduced with lentivirus encoding shRNA to HIF-1α or Scrambled (Scr) RNA and stable cell lines were generated with puromycin selection. Total RNA and nuclear protein extracts were extracted from the cells to confirm the status of the knockdown. (<b>B</b>) mRNA levels of HIF-1α measured by RT-qPCR after 48 hours in normoxia(N) or hypoxia(H). mRNA levels are normalized to that of 18S ribosomal RNA and are expressed as fold change relative to cells containing shScr under normoxia. (<b>C and D</b>) Protein levels of HIF-1α measured by Western blot analysis of nuclear extracts after 24 hours in culture. β-actin is shown as a loading control. (<b>C</b>) Normoxic levels of HIF-1α levels in the absence or presence of 50μM cobalt chloride (CoCl₂), a hypoxia mimic that prevents oxygen-induced degradation of HIF-1α. (<b>D</b>) HIF-1α levels under normoxia or hypoxia.</p

Effects of HIF-1α inhibitor PX-478 on PELs.

<p>(<b>A</b>) HIF-1α mRNA levels in BCBL-1 cells 24 hours after treatment with various concentrations of PX-478, normalized to 18S internal control and expressed as fold changes compared to no PX-478 control cells. (<b>B)</b> Proliferation rate of PEL cell lines BCBL-1, BC-3, JSC-1, BC-1, and BC-2 and Burkitt’s lymphoma (BL) cell lines BJAB and CA46 measured using the MTS assay 72 hours after treatment with indicated concentrations of PX-478, expressed as fold changes compared to no PX-478 control cells. (<b>C)</b> Growth rates of the PEL and BL cells treated with 0 or 10 μM PX-478. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between untreated and inhibitor-treated cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01.</p

Effect of HIF-1α knockdown on the expression of KSHV miRNAs.

<p><b>(A)</b> Level of primary miRNA transcript as measured by RT-qPCR and normalized to 18S mRNA. <b>(B)</b> Levels of mature miRNAs measured using taqman assays and normalized to that of RNU43 miRNA. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05 (2-tailed t-test).</p

Effect of HIF-1α knockdown on the expression of KSHV latent genes in PELs.

<p>(<b>A</b>) Schematic of multicistronic KSHV latency locus showing the location of latent genes and hypoxia-response elements (HREs) in the promoter region. (<b>B</b>) mRNA levels of latent genes in BCBL-1 cells within and (<b>C</b>) outside of the latency locus as measured by RT-qPCR and normalized to that of 18S RNA. RNA levels for each gene are expressed as fold change relative to shScr cells under normoxia (N). Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01 (2-tailed t-test).</p

HIF-1α knockdown leads to reduced lytic replication of KSHV.

<p><b>(A)</b> mRNA levels of RTA, vIL-6, and ORF26 (a late lytic gene) of KSHV in BCBL-1 cells measured by RT-qPCR. Error bars represent standard deviation from at least 3 experiments. (<b>B)</b> Western blot showing protein levels of intracellular RTA, vIL6, and β-actin in the cell lysates as well as secreted vIL6 in the supernatant after 48 hours in normoxia (N) or hypoxia (H). (<b>C)</b> Protein levels of ORF45 in concentrated virus particles released in the supernatants after 72 hours. *Unspecific band. Western blots were done on lysates from three independent experiments and representative blots are shown.</p