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

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

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

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

Toward Eradicating HIV Reservoirs in the Brain: Inhibiting P-Glycoprotein at the Blood–Brain Barrier with Prodrug Abacavir Dimers

Eradication of HIV reservoirs in the brain necessitates penetration of antiviral agents across the blood–brain barrier (BBB), a process limited by drug efflux proteins such as P-glycoprotein (P-gp) at the membrane of brain capillary endothelial cells. We present an innovative chemical strategy toward the goal of therapeutic brain penetration of the P-gp substrate and antiviral agent abacavir, in conjunction with a traceless tether. Dimeric prodrugs of abacavir were designed to have two functions: inhibit P-gp efflux at the BBB and revert to monomeric therapeutic within cellular reducing environments. The prodrug dimers are potent P-gp inhibitors in cell culture and in a brain capillary model of the BBB. Significantly, these agents demonstrate anti-HIV activity in two T-cell-based HIV assays, a result that is linked to cellular reversion of the prodrug to abacavir. This strategy represents a platform technology that may be applied to other therapies with limited brain penetration due to P-glycoprotein

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

Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

<div><p>Kaposi’s sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi’s sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.</p></div

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