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

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

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

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 suppression on growth of PELs.

<p><b>(A)</b> Growth rate of shScr or shHIF-1 BCBL-1 and BC-3 cells measured by counting live cells every 24 hours in normoxia. (<b>B)</b> Proliferation rate of shScr or shHIF-1 cells measured by MTS assay at 72 hours in normoxia using Promega’s CellTiter 96 Aqueous One Solution assay, which measures the amount of NADH or NADPH produced by metabolically active cells. (<b>C)</b> Colony forming efficiency of shHIF-1 cells relative to shScr cells. Error bars represent standard deviations from at least 3 independent experiments. Results shown in B and C are fold changes compared to shScr cells. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01.</p