The effect of H1.2 and Aly on EPHX1 promoter activity.

<p>A. EPHX1 promoter activity using the -1797/+3460 construct (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125318#pone.0125318.g003" target="_blank">Fig 3</a>) was measured in HepG2 cells expressing H1.2 (lane 2,5), ALY (lane 3) and H1.2 and ALY (lanes 4,6). B. The effect of H1.2 shRNA on EPHX1 promoter activity. Values are the mean +/- S.D. of independent experiments (n = 3–4) performed in triplicate. C. Western blot of H1.2 levels following treatment with 0.7 μg of H1.2 shRNA.</p

Characterization of the interaction of PARP-1 with the -20/-10 EPHX1 promoter region.

<p>A. EMSA analysis was performed with a HepG2 nuclear protein extract and ³²P-labeled oligonucleotides (-28/-4) containing the WT PARP-1 sequence in the absence and presence of a 50-fold excess of unlabeled oligonucleotide WT competitor (lane 2,1), and in the presence of a 50-fold excess of an oligonucleotide (Px) of unrelated sequence that can also binds PARP-1 (lane 3). EMSA was performed with the labeled -17 mutated oligonucleotide (lane 4). Nuclear extracts were preincubated with a PARP-1 antibody (lane 5,6) or a GATA-6 antibody (lane 7,8) prior to EMSA analysis. B. The effect of PARP-1 shRNA on the promoter activity of the EPHX1 -8-/+25 construct. Values are the mean +/- S.D. of independent experiments (n = 3–5) performed in triplicate. C. Western blot of PARP-1 levels following treatment with 0.4 μg PARP-1 shRNA. D. The interaction of PARP-1 with the EPHX1 proximal promoter as assessed by ChIP analysis. Crosslinked protein-DNA complexes were incubated with anti-PARP-1 antibody, isolated by protein A-Sepharose beads and DNA corresponding to the EPHX1–297/+25 and EPHX1 +2462/+2641 which does not contain the PARP-1 binding site were analyzed by PCR. The band observed for PARP-1 in the EPHX1 promoter region (lane 2). Immunoprecipatation with IgG (lane 1). PCR analysis with primers to the +2462/+2641 region (lane 3). Gels were scanned and quantitated using UN-SCAN-IT gel automated digitizing system. Values are the result of 3 independent experiments.</p

Analysis of EPHX1 transcriptional activity mediated by the proximal promoter region in HepG2 cells.

<p>A: Map of the EPHX1 promoter indicating the location of 3 naturally occurring T>C mutations observed in hypercholanemic subject 1d [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125318#pone.0125318.ref021" target="_blank">21</a>]. B: The effect of the T>C mutations on EPHX1 promoter activity. WT and M forms of the -80/+25 EPHX1 promoter region were inserted into the pGL3 expression vector containing the luciferase reporter gene (LUC). Values are the mean +/- S.D. of independent experiments (n = 3–4) performed in triplicate. <i>P</i><0.01 for all studies reported. C: Nucleotide sequence of the EPHX1 proximal promoter (-80/+1) indicating the location of the critical -17 T>C substitution within the PARP-1 binding site as well as neighboring transcription factor binding sites previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125318#pone.0125318.ref016" target="_blank">16</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125318#pone.0125318.ref018" target="_blank">18</a>]. D: The effect of the -17 mutation on EPHX1 promoter activity driven by the -80/+25 promoter.</p

Characterization of the interaction of H1.2 with the EPHX1 intron 1 region.

<p>A. Map showing the EPHX1 promoter indicating the -17 T>C mutation and the location of the intron 1 polymorphism at +2557 and the sequence of the oligonucleotide used to isolate the H1.2/Aly complex. B. The effect of the C>G at +2557 on EPHX1 transcription using the -1797/+3460 fragment in the pGL3 vector containing the luciferase reporter gene. Values are the mean +/-S.D. of independent experiments (n = 3–5) performed in triplicate C. DNA affinity chromatography in conjunction with streptavidin-coated magnetic beads using the WT (lane 1) and C>G mutated (lane 2) 100 bp oligonucleotides (+2508/+2607) analyzed by SDS-PAGE and quantitated using the UN-SCAN-IT gel automated digitizing system. The bands were cut from the gel and identified by mass spectrometry. D. Comparison of the sequences in the PARP-1 and H1.2 binding sites.</p

Effect of different fractions against aduit Schistosoma japonicum worms in vitro.

a<p>RPMI 1640 medium and 1% DMSO in RPMI 1640 medium were used as negative control groups.</p>b<p>Praziquantel (PQZ, 30 ug/ml) was used as positive control groups. Data are presented as the mean of four experiments.</p>c<p>Number of worms tested.</p>1<p>Drug group compared with RPMI 1640, 1%DMSO negative control group, both P values<0.05;</p>2<p>Drug group compared with PZQ positive group, both P values<0.05;</p

PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription-3

<p><b>Copyright information:</b></p><p>Taken from "PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription"</p><p>http://www.jmolecularsignaling.com/content/2/1/5</p><p>Journal of Molecular Signaling 2007;2():5-5.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC1939705.</p><p></p>porter plasmid, pArg maxi, along with 20 nM of either siRNA specific for PNRC (PNRC/si) or nonspecific mismatch RNA (mm). Twenty-four hours after transfection, 20 μg of total RNA, isolated from the transfected cells, was subjected to Northern analysis with PNRC and GAPDH cDNA probes, separately, as described in Methods section. , Western blot of PNRC and actin protein. An aliquot of cell lysate equal to 100 μg of protein prepared from PNRC/siRNA (20 nM) transfected (PNRC/si) or nonspecific mismatch RNA (mm) transfected MCF7/EGFP-PNRC cells was separated on a 10% SDS-PAGE and subjected to Western analysis with 1:1000 diluted anti-GFP mouse monoclonal antibody (Cloetech) and anti-actin antibody (Santa Cruz Biotechnology) separately. The protein bands with molecular weights corresponding to those of EGFP-PNRC fusion protein or actin were indicated by arrows. , Down regulation of tRNAarg transcription by PNRC/siRNA. MCF-7 cells were transiently transfected with reporter plasmid, pArg maxi (10 μg) along with 20 nM of either PNRC/siRNA (PNRC) or mismatch control RNA (mm). Twenty-four hours after transfection, the levels of tRNA transcripts in the transfected cells were determined by RNase protection assay as described in figure 3

PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription-5

<p><b>Copyright information:</b></p><p>Taken from "PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription"</p><p>http://www.jmolecularsignaling.com/content/2/1/5</p><p>Journal of Molecular Signaling 2007;2():5-5.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC1939705.</p><p></p>BD-PNRCas bait. To confirm the interaction and the specificity of the interaction between PNRC and RPC39, yeast strain Y187 was cotransformed with the expression plasmids for the expression of the fusion proteins as indicated, and transformants containing these plasmids were selected by growth on SD/-Leu/-Trp agar plates. The expression of interacting hybrid proteins in Y187 transformants was analyzed for Z expression. DBD(DBD) and DBD-human lamin C (hLC) expression plasmids were included as background and negative control, respectively. β-Galactosidase activities in liquid cultures are expressed in Miller units as mean ± s.d. of three independent assays. , PNRC interacts with the C-terminus, amino acids 212–316, of the human Pol III subunit RPC39. The yeast expression plasmids, pACT2-RPC39 and pACT2-RPC39, for the expression of AD-RPC39 wild type (WT) or AD-RPC(1–212) fusion proteins were constructed as described in 'Methods'. The expression plasmids for AD-RPC39(212–316) and AD-RPC39(241–316) were isolated from library screening through their interaction with PNRC. Y187 cells were cotransformed with the expression plasmids coding for the fusion proteins as indicated. The selection of Y187 transformants and the β-Gal assays on transformants were performed as described in

Effect of BE-IS on the viability of RAW264.7 cells.

<p>Cells were incubated for 24% DMSO, 1 µg/ml LPS, or BE-IS (1.25, 2.5, 5, 10, 20 µg/ml, dissolved in 0.1% DMSO). Cell viability was determined by MTT assay. The optical density was measured at 550 nm on a microplate reader. Values are expressed as mean ± S.E. for three different experiments performed in triplicate.</p

PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription-1

<p><b>Copyright information:</b></p><p>Taken from "PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription"</p><p>http://www.jmolecularsignaling.com/content/2/1/5</p><p>Journal of Molecular Signaling 2007;2():5-5.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC1939705.</p><p></p>on plasmid, pSG5-RPC39, 24 h post-transfection, cells were harvasted and lysed, and 15 mg of total proteins were immunoprecipitated with antibodies against GFP (Clontech) or RPC39 (Santa Cruz Biotechnology). Specifically bound proteins to Protein A agarose beads were separated on a 10% SDS-PAGE and analysed by immunoblotting with either anti-RPC39 (, blot: anti-RPC39) or anti-GFP (, blot: anti-GFP) antibody, as previously described [2]. An aliquot of cell lysate equal to 100 μg of protein was included in each SDS-PAGE gel for Western blot to examine whether the crude protein extracts used for co-immunoprecipitation contains EGFP-PNRC and RPC39 proteins. The protein bands with molecular weights corresponding to those of RPC39 or EGFP-PNRC were indicated by arrows

Chemical composition of the PE fraction identified by GC-MS and Kovats Index.

a<p>Retention times (minute).</p>b<p>Retention index was calculated from our analyses with respect to a series of n-alkenes.</p>c<p>Percentage of extracted amount to total.</p