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

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

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

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

<i>In vitro</i> antiviral activities of fangchinoline against different HIV-1 laboratory strains.

<p>Antiviral assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039225#s2" target="_blank">Materials and Methods</a>. Values represent average of at least three independent experiments.</p>a<p>Compound concentration required to reduce the production of p24 antigen by 50%.</p>b<p>Compound concentration required to reduce mock-infected cell viability by 50%.</p>c<p>Selective index, ratio of CC₅₀/EC₅₀.</p

Anti-HIV activities of test compounds in MT-4 CPE assays and TZM-b1 assays.

<p>Antiviral assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039225#s2" target="_blank">Materials and Methods</a>. Values represent average of at least three independent experiments.</p>a<p>Compound concentration required to reduce the virus-induced CPE by 50%.</p>b<p>Compound concentration required to reduce the luciferase expression in virus infected TZM-b1 cells by 50%.</p

Fangchinoline inhibits HIV-1 NL4-3 replication in MT-4 cells.

<p>(A and B) MT-4 cells were infected with HIV-1 NL4-3 at an MOI of 0.02 or mock infected in the presence of serially diluted fangchinoline. On day 4 post-infection, compound cytotoxicity was determined in parallel in mock-infected cells (A), and the level of viral replication in infected cells was determined by p24 antigen capture ELISA (B). The results were presented as the mean values with standard deviations. (C and D) MT-4 cells were infected with HIV-1 NL4-3 at an MOI of 0.02 in the presence of test compounds (AZT, 0.05 µM; fangchinoline, 0.3–2.5 µM), and genomic DNA and mRNA from infected cells were isolated 3 days post-infection. Total viral DNA (C, upper panel), integrated proviral DNA (C, middle panel) were examined by semi-quantitative PCR analysis. Viral mRNA levels were examined by semi-quantitative reverse transcription PCR analysis (D, upper panel). GAPDH was used as both the DNA and RNA input control.</p