Table_1_Influenza A virus NS1 protein represses antiviral immune response by hijacking NF-κB to mediate transcription of type III IFN.docx

BackgroundNon-structural protein 1 (NS1), one of the viral proteins of influenza A viruses (IAVs), plays a crucial role in evading host antiviral immune response. It is known that the IAV NS1 protein regulates the antiviral genes response mainly through several different molecular mechanisms in cytoplasm. Current evidence suggests that NS1 represses the transcription of IFNB1 gene by inhibiting the recruitment of Pol II to its exons and promoters in infected cells. However, IAV NS1 whether can utilize a common mechanism to antagonize antiviral response by interacting with cellular DNA and immune-related transcription factors in the nucleus, is not yet clear.MethodsChromatin immunoprecipitation and sequencing (ChIP-seq) was used to determine genome-wide transcriptional DNA-binding sites for NS1 and NF-κB in viral infection. Next, we used ChIP-reChIP, luciferase reporter assay and secreted embryonic alkaline phosphatase (SEAP) assay to provide information on the dynamic binding of NS1 and NF-κB to chromatin. RNA sequencing (RNA-seq) transcriptomic analyses were used to explore the critical role of NS1 and NF-κB in IAV infection as well as the detailed processes governing host antiviral response.ResultsHerein, NS1 was found to co-localize with NF-κB using ChIP-seq. ChIP-reChIP and luciferase reporter assay confirmed the co-localization of NS1 and NF-κB at type III IFN genes, such as IFNL1, IFNL2, and IFNL3. We discovered that NS1 disturbed binding manners of NF-κB to inhibit IFNL1 expression. NS1 hijacked NF-κB from a typical IFNL1 promoter to the exon-intron region of IFNL1 and decreased the enrichment of RNA polymerase II and H3K27ac, a chromatin accessibility marker, in the promoter region of IFNL1 during IAV infection, consequently reducing IFNL1 gene expression. NS1 deletion enhanced the enrichment of RNA polymerase II at the IFNL1 promoter and promoted its expression.ConclusionOverall, NS1 hijacked NF-κB to prevent its interaction with the IFNL1 promoter and restricted the open chromatin architecture of the promoter, thereby abating antiviral gene expression.</p

WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

<div><p>Renal cell carcinoma (RCC) is a malignancy with poor prognosis. WNT/β-catenin signaling dysregulation, especially β-catenin overactivation and WNT antagonist silencing, is associated with RCC carcinogenesis and progression. However, the role of WNT ligands in RCC has not yet been determined. We screened 19 WNT ligands from normal kidney and RCC cell lines and tissues and found that WNT10A was significantly increased in RCC cell lines and tissues as compared to that in normal controls. The clinical significance of increase in WNT10A was evaluated by performing an immunohistochemical association study in a 19-year follow-up cohort comprising 284 RCC and 267 benign renal disease (BRD) patients. The results of this study showed that WNT10A was dramatically upregulated in RCC tissues as compared to that in BRD tissues. This result suggests that WNT10A, nuclear β-catenin, and nuclear cyclin D1 act as independent risk factors for RCC carcinogenesis and progression, with accumulative risk effects. Molecular validation of cell line models with gain- or loss-of-function designs showed that forced WNT10A expression induced RCC cell proliferation and aggressiveness, including higher chemoresistance, cell migration, invasiveness, and cell transformation, due to the activation of β-catenin-dependent signaling. Conversely, WNT10A siRNA knockdown decreased cell proliferation and aggressiveness of RCC cells. In conclusion, we showed that WNT10A acts as an autocrine oncogene both in RCC carcinogenesis and progression by activating WNT/β-catenin signaling.</p> </div

Estrogen Enhances the Cell Viability and Motility of Breast Cancer Cells through the ERα-ΔNp63-Integrin β4 Signaling Pathway

<div><p>Estrogen induces ERα-positive breast cancer aggressiveness via the promotion of cell proliferation and survival, the epithelial-mesenchymal transition, and stem-like properties. Integrin β4 signaling has been implicated in estrogen/ERα-induced tumorigenicity and anti-apoptosis; however, this signaling cascade poorly understood. ΔNp63, an N-terminally truncated isoform of the p63 transcription factor, functions as a transcription factor of integrinβ4 and therefore regulates cellular adhesion and survival. Therefore, the aim of the present study was to investigate the estrogen-induced interaction between ERα, ΔNp63 and integrin β4 in breast cancer cells. In ERα-positive MCF-7 cells, estrogen activated ERα transcription, which induced ΔNp63 expression. And ΔNp63 subsequently induced integrin β4 expression, which resulted in AKT phosphorylation and enhanced cell viability and motility. Conversely, there was no inductive effect of estrogen on ΔNp63-integrinβ4-AKT signaling or on cell viability and motility in ERα-negative MDA-MB-231 cells. ΔNp63 knockdown abolishes these estrogen-induced effects and reduces cell viability and motility in MCF-7 cells. Nevertheless, ΔNp63 knockdown also inhibited cell migration in MDA-MB-231 cells through reducing integrin β4 expression and AKT phosphorylation. In conclusion, estrogen enhances ERα-positive breast cancer cell viability and motility through activating the ERα-ΔNp63-integrin β4 signaling pathway to induce AKT phosphorylated activation. Those findings should be useful to elucidate the crosstalk between estrogen/ER signaling and ΔNp63 signaling and provide novel insights into the effects of estrogen on breast cancer progression.</p></div

WNT10A increases colony formation ability.

<p>Colony formation assay was used to evaluate the effect of WNT10A on cell transformation. After 48 h of transfection, 2×10⁴ cells were mixed with 0.3% agarose in 1× complete RPMI and transferred into a coated 6-cm dish, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047649#s2" target="_blank">Materials and Methods</a>. To prolong the effects of transfection, pcDNA-WNT10A-transfected cells were maintained in 200 µg/mL G418, and WNT10A siRNA or β-catenin siRNA knockdown cells were maintained in 10 nM of WNT10A siRNA for 15 days. Forced WNT10A expression in 786-O significantly increased the number of colonies of these cells; cotransfection with β-catenin siRNA reduced the WNT10A promotive effect on colony formation ability. Moreover, each WNT10A siRNA knockdown in Caki-1 reduced the number of colonies of these cells. Bar charts show the number of colonies; significances were analyzed by using the Student’s <i>t</i>-test.</p

Univariate and multivariate analyses of prognostic factors and RCC survival.

*<p>Categorized as low (≤mean) and high (>mean) based on the values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047649#pone-0047649-g002" target="_blank">Figure 2B</a>.</p>†<p>Analyzed with Cox harzard regression model, and the statistic significance (p<0.05) is showed in boldfaced. (n.a.: not analyzed).</p

Expression of 19 <i>WNT</i> genes in kidney cell lines and tissues.

<p>(A) mRNA expression profiles of <i>WNT</i> genes from kidney cell lines obtained using RT-PCR. mRNA expression profiles of 19 <i>WNT</i> genes from 5 RCC cell lines (786-O, Caki-1, RCC-1, A498, and ACHN) and 1 immortalized proximal tubule epithelial cell line from a normal adult human kidney (HK-2) were obtained using RT-PCR. Higher WNT10A expression was observed in RCC cell lines Caki-1, RCC-1, and ACHN, but lower expression was observed in RCC cell lines 786-O and A498. WNT10A expression was undetectable in the normal kidney cell line HK-2. (B) mRNA expression profile of <i>WNT</i> genes from BRD and RCC specimens obtained using RT-PCR. mRNA expression profiles of 19 <i>WNT</i> genes from 6 paired RCC (T1–T6) and paratumoral (N1–N6) tissues. mRNA expression profiles of <i>WNT</i> genes from other 4 BRD tissues (N7–N10) were also examined. Higher expression of WNT10A was observed in most RCC tissues than in paratumoral and BRD tissues. (C) Quantitative real-time PCR for detecting the expression of <i>WNT</i> genes. Expression of <i>WNT</i> genes in each cell line was determined using SYBR Green real-time PCR. An independent data set comprising 10 RCC and 10 normal kidney tissues was used. The results obtained were similar to those obtained for RT-PCR, i.e., higher expression of WNT10A was observed in most RCC cell lines and tissues than in normal controls. All experiments were performed in triplicate. Relative expression of each gene from each cell line was normalized with that of the gene from HK-2 normal kidney cell line; relative expression of each gene of tissues was normalized with the mean of 10 normal kidney tissues.</p

Mechanistic Investigation of Oxidative Decarboxylation Catalyzed by Two Iron(II)- and 2‑Oxoglutarate-Dependent Enzymes

Two non-heme iron enzymes, IsnB and AmbI3, catalyze a novel decarboxylation-assisted olefination to produce indole vinyl isonitrile, an important building block for many natural products. Compared to other reactions catalyzed by this enzyme family, decarboxylation-assisted olefination represents an attractive biosynthetic route and a mechanistically unexplored pathway in constructing a CC bond. Using mechanistic probes, transient state kinetics, reactive intermediate trapping, spectroscopic characterizations, and product analysis, we propose that both IsnB and AmbI3 initiate stereoselective olefination via a benzylic C–H bond activation by an Fe­(IV)–oxo intermediate, and the reaction likely proceeds through a radical- or carbocation-induced decarboxylation to complete CC bond installation

Forced WNT10A expression induced higher chemoresistance in 786-O, and WNT10A siRNA knockdown reduced chemoresistance in Caki-1.

<p>Effects of WNT10A on cell survival were analyzed using PI staining with flow cytometry. pcDNA-WNT10A-transfected 786-O showed slight increment in G1 phase as compared to that observed in vector-transfected controls after 48 h of solvent (DMSO) treatment. Moreover, cotransfection of pcDNA-WNT10A and β-catenin siRNA showed an obvious G1 arrest (left lane of 786-O). However, pcDNA-WNT10A-transfected 786-O showed lower sub-G1 cell population than vector-transfected controls after 48 h of treatment with 2 µM epirubicin (middle lane of 786-O) or 10 µM cisplatin (right lane of 786-O). Besides, cotransfection of pcDNA-WNT10A and β-catenin siRNA increased the chemosensitivity of 786-O. Conversely, WNT10A siRNA-transfected Caki-1 showed minor sub-G1 and G1 phase modifications as compared to those observed in scrambled siRNA-transfected controls. However, β-catenin siRNA induced both higher sub-G1 and G1 arrest (left lane of Caki-1). WNT10A siRNA- and β-catenin siRNA-transfected Caki-1 showed significantly increase sub-G1 cell population than scrambled siRNA-transfected controls after 48 h of treatment with 2 µM epirubicin (middle lane of Caki-1) or 10 µM cisplatin (right lane of Caki-1). The proportion of each cell cycle phase belonging to both cell lines are shown using bar charts.</p

Forced WNT10A expression and WNT10A siRNA knockdown in kidney cell lines.

<p>(A) Cell growth curve of pcDNA-WNT10A-transfected cells and WNT10A siRNA knockdown cells. WNT10A gain-of-function was achieved by transfecting pcDNA-WNT10A in normal kidney cell line HK-2 and RCC cell lines 786-O and A498, which had relatively lower endogenous WNT10A expression. Cotransfection of pcDNA-WNT10A and β-catenin siRNA was also performed. Conversely, WNT10A loss-of-function was achieved by WNT10A siRNA knockdown in RCC-1 and Caki-1, which have higher endogenous WNT10A. After transient transfection for 48 h, cell viability was determined using the MTT assay and was compared with that of cells transfected with Lipofectamine only (reagent control), pcDNA3.1 vector only (vector), and pcDNA-WNT10A and with that of cells cotransfected with pcDNA-WNT10A and β-catenin siRNA at 12, 24, 48, and 72 h. HK-2, 786-O, and A498 transfected with pcDNA-WNT10A showed significant increase in cell proliferation after 48 h; however, cotransfection of pcDNA-WNT10A and β-catenin siRNA reduced WNT10A-induced cell proliferation. Conversely, RCC-1 and Caki-1 transfected with WNT10A siRNA showed significant decrease in cell proliferation as compared to cells transfected with reagent and scrambled siRNA controls. (**<i>P</i><0.001, *<i>P</i><0.05; Student’s <i>t</i>-test). Western blot analysis of pcDNA-WNT10A- and WNT10A siRNA-transfected cells from each cell line was also performed. HK-2, 786-O, and A498 were transfected with 3 µg of pcDNA-WNT10A, pcDNA3.1 vector alone, and cotransfected with β-catenin siRNA for 48 h. Conversely, RCC-1 and Caki-1 were transfected with 1 µg WNT10A siRNA or scrambled siRNA controls for 72 h. Twenty micrograms of total protein extract from each cell line was loaded onto SDS-polyacrylamide gel and western blot analysis was performed. Forced WNT10A expression in HK-2, 786-O, and A498 remarkably increased the concentration of WNT10A than that of the vector control in these cells. Forced WNT10A expression also upregulated nuclear β-catenin, cyclin D1, and c-myc levels, and cotransfection with β-catenin siRNA reduced cyclin D1 and c-myc expression levels. Conversely, WNT10A siRNA knockdown in RCC-1 and Caki-1 markedly reduced endogenous WNT10A levels, thus reducing nuclear β-catenin, cyclin D1, and c-myc levels. (B) Immunocytochemical analysis of pcDNA-WNT10A-transfected cells and WNT10A siRNA knockdown cells. After pcDNA-WNT10A was transfected in HK-2 and 786-O, expression of WNT10A, β-catenin, and cyclin D1 was determined by immunocytochemistry. WNT10A levels were significantly increased in the transfected cells. β-catenin showed high intracellular accumulation in WNT10A-transfected cells and low membranous expression in vector- and reagent-transfected controls. Moreover, cyclin D1 was upregulated in the nucleus of pcDNA-WNT10A-transfected cells but showed low cytoplasmic expression in vector- and reagent-transfected controls. Upregulated nuclear β-catenin and cyclin D1 were also observed in the same pcDNA-WNT10A-transfected cells (red staining, β-catenin; brown staining, cyclin D1). Conversely, WNT10A siRNA-transfected RCC-1 and Caki-1 showed an obvious reduction in endogenous WNT10A expression and reduced intracellular β-catenin accumulation and cyclin D1 expression. (C) TCF/LEF reporter assay. Forced WNT10A expression in 786-O for 48 h significantly induced luciferase activity in these cells than that in vector- and reagent-transfected controls. Conversely, WNT10A siRNA-transfected Caki-1 showed significantly reduced luciferase activity after 72 h as compared to that in vector- and reagent-transfected controls.</p

Effects of estrogen on ΔNp63 and TAp63 expression in MCF-7 and MDA-MB-231 cells.

<p>MCF-7 and MDA-MB-231 cells were cultured in 10% FBS/DMEM with 10nM estrogen or 0.1% ethanol for 0, 1, 2, 4, and 8 h, and protein and mRNA expression was detected by (A) western blotting and real-time RT-PCR for (B) ΔNp63 and (C) TAp63. In MCF-7, ΔNp63 expression peaked at 2 h after estrogen treatment at both the protein and mRNA levels, and estrogen treatment did not significantly affect TAp63 protein and mRNA expression levels. In MDA-MB-231, estrogen treatment did not affect ΔNp63 and TAp63 expression in both protein and mRNA levels. All data are the mean ± SD of triplicate experiments.</p