KSHV-infected cells show impaired response to IL-22 stimulation.

<p>293T and 293T-BAC36 cells are stimulated with 100 ng/ml of rIL-22 for 0, 5, 15, or 30 min. The cells were lysed and subjected to immunoblot analysis to detect the phosphorylation of STAT3 and ERK.</p

Sequences of the primers used for the generation of luciferase reporter plasmids.

<p>Restriction enzyme sites are underlined.</p

Expression levels of cytokines and cytokine receptors in KS lesions vs. normal tissues.

<p>A cDNA microarray analysis was performed to compare the gene expression between KS tissue and normal tissue. Differentially expressed interleukin-associated genes are listed (Ratio ≥2 or ≤0.5 and p value of log-ratio <0.05).</p

The LBS-like sequence is required for LANA to down-regulate IL-22R1 expression.

<p>(<b>A</b>) Schematic of pIL22R1 (−2139 to +39), pIL22R1ΔLBS-like and pIL22R1mLBS-like DNA sequences. (<b>B</b>) Increasing amounts of pcDNA-LANA expressing full-length LANA were co-transfected with either pIL22R1 (−2139 to +39) or its mutants (pIL22R1ΔLBS-like and pIL22R1mLBS-like) into 293T cells. At 48 h post-transfection, cells were harvested and assayed for luciferase activity. (<b>C</b>) Increasing amounts of pcDNA-LANA-C-expressing carboxyl-terminal domain (amino acids 951–1162) of LANA were co-transfected with either pIL22R1 (−2139 to +39) or its mutants (pIL22R1ΔLBS-like and pIL22R1mLBS-like) into 293T cells. At 48 h post-transfection, cells were harvested and assayed for luciferase activity. (<b>D</b>) Increasing amounts of pcDNA-LANA expressing full-length LANA were co-transfected with either pIL22R1 (−2139 to +39) or its mutants (pIL22R1ΔLBS-like and pIL-22R1mLBS-like) into HUVEC cells. At 48 h post-transfection, cells were harvested and assayed for luciferase activity.</p

LANA binds to the IL-22R1 promoter in vitro.

<p>(<b>A</b>) The aligned sequences of DNA fragments from TR DNA, wild type and mutant IL-22R1 promoters which contain LBS, the wild type LBS-like sequence and scrambled-mutant DNA sequence (underlined) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019106#pone.0019106-Garber2" target="_blank">[38]</a>. The nucleotide sequences represent the probe sequences used in the EMSA and were labeled with biotin. (<b>B</b>) LANA can bind to the LBS-like DNA sequence in IL-22R1 promoter. Probes as indicated were incubated for 20 min with or without purified LANA-C (a.a. 951–1162). NC, a DNA fragment derived from the sequence in M13 DNA, used as a negative control for LANA binding. (<b>C</b>) The binding of LANA to the wild type LBS-like sequence is specific. A 10-fold excess of cold competitor or mutant competitor DNA was added to compete the reaction between WT LBS-like and LANA. The upper and lower arrows indicate the LANA-specific binding band and free probe.</p

LANA down-regulates IL-22R1 promoter activity in a dose-dependent manner.

<p>(<b>A</b>) Schematic of pIL22R1 (−2139 to +39) and a series of deletion mutants (pIL22R1-D1, D2, D3, and D4). (<b>B</b>) LANA down-regulates IL-22R1 promoter (−2139 to +39) activity. pIL22R1 (−2139 to +39) was co-transfected with increasing amounts (0, 0.1, 0.2 or 0.4 µg) of LANA into 293T cells. At 48 h post-transfection, cells were lysed and assayed for luciferase activity. The expression levels of LANA were detected in cell lysates by western blotting using anti-LANA-C antibody (top panel). The regulation of the IL-22R1 promoter (−2139 to +39) by LANA was also analyzed in HUVEC cells (lower panel).</p

LANA interacts with the IL-22R1 promoter in vivo.

<p>(<b>A</b>) Schematic diagram showing the locations of the two pairs of primers used in the ChIP assay. (<b>B</b>) Formaldehyde cross-linked chromatin was prepared from 293T cells that were transfected with pFLAG, pFLAG-LANA, pFLAG-LANA_1–939 or pFLAG-LANA_933–1162, and immunoprecipitated with anti-FLAG antibody. PCR was performed with primer pair 1 to amplify a 197 bp DNA fragment containing LBS or with primer pair 2 to amplify a 283 bp DNA fragment, approximately 2 kb upstream of LBS-like sequence in IL-22R1 promoter. A sample representative of 5% the total input chromatin was included in the PCR analysis. (<b>C</b>) The expression levels of LANA and its truncated mutants were detected in cell lysates by western blotting using anti-FLAG antibody and anti-actin served as a loading control.</p

Conserved domains of MdLBDs gene family.

<p>A. The CX₂CX₆CX₃C zinc finger-like domain sequence logos. B. The LX6LX3LX6L leucine zipper-like domain sequences Logos. Sequence alignment of two domains by ClustalX and conserved motifs Logos was performed by the WebLogo program.</p

A Genome-Wide Analysis of the LBD (LATERAL ORGAN BOUNDARIES Domain) Gene Family in <em>Malus domestica</em> with a Functional Characterization of <em>MdLBD11</em>

<div><p>The plant-specific LBD (LATERAL ORGAN BOUNDARIES domain) genes belong to a major family of transcription factor that encode a zinc finger-like domain. It has been shown that LBD genes play crucial roles in the growth and development of <i>Arabidopsis</i> and other plant species. However, no detailed information concerning this family is available for apple. In the present study, we analyzed the apple (<i>Malus domestica</i>) genome and identified 58 <i>LBD</i> genes. This gene family was tested for its phylogenetic relationships with homologous genes in the <i>Arabidopsis</i> genome, as well as its location in the genome, structure and expression. We also transformed one <i>MdLBD</i> gene into <i>Arabidopsis</i> to evaluate its function. Like <i>Arabidopsis</i>, apple <i>LBD</i> genes also have a conserved CX₂CX₆CX₃C zinc finger-like domain in the N terminus and can be divided into two classes. The expression profile indicated that apple <i>LBD</i> genes exhibited a variety of expression patterns, suggesting that they have diverse functions. At the same time, the expression analysis implied that members of this apple gene family were responsive to hormones and stress and that they may participate in hormone-mediated plant organogenesis, which was demonstrated with the overexpression of the apple <i>LBD</i> gene <i>MdLBD11</i>, resulting in an abnormal phenotype. This phenotype included upward curling leaves, delayed flowering, downward-pointing flowers, siliques and other abnormal traits. Based on these data, we concluded that the <i>MdLBD</i> genes may play an important role in apple growth and development as in <i>Arabidopsis</i> and other species.</p> </div

Tissue-specific expression profiles and gene response for the <i>MdLBD</i> genes.

<p>A. Tissue-specific expression profiles of <i>MdLBD</i> genes. Expression levels of <i>MdLBD</i> genes were examined by semi-qRT-PCR in apple roots (R), stems (S), leaves (L), flowers (FL) and fruits (F). The <i>MdACTIN</i> was performed as an internal control. B. QRT-PCR analysis of <i>MdLBD</i> genes in response to multiple treatments. <i>MdLBD</i> genes expression treated by 15 mM KNO₃ (15 mM KCl was treated as a control), hypoxia, and 100 µM of ABA, NAA, 6-BA, GA and BR, the <i>MdACTIN</i> was performed as an internal control.</p