LPS treatment regulates CD274 mRNA levels mainly via the NF-κB signaling pathway in primary human monocytes.

<p>CD274 mRNA levels were determined by quantitative real-time PCR and are shown as fold changes in arbitrary units. (A) LPS treatment regulates CD274 mRNA levels (n = 3, repeated three times, *P<0.05 vs. control without LPS treatment). (B) Inhibitory effects of BAY (an NF-κB inhibitor), T-5224 (an AP-1 inhibitor), Wort (a PI3K inhibitor) and DMSO on CD274 mRNA levels after LPS treatment (n = 3, repeated three times, *P<0.05 vs. control with LPS treatment for 12 h). (C) Inhibitory effects of BAY, T-5224, Wort and DMSO on CD274 protein levels after LPS treatment for 24 h, representative western blot evaluating cytoplasmic CD274 protein expression from primary human monocytes. (D) Time kinetics of nuclear translocation of NF-κB after LPS treatment. Representative western blot evaluating p65 protein levels in nuclear and cytoplasmic proteins from primary human monocytes. IκB-α protein levels in cytoplasmic proteins indirectly indicates nuclear translocation of NF-κB. LPS was added to cells and incubated for 0, 0.5, 1, 1.5 and 2 h.</p

LPS and p65 siRNA combined treatment regulates CD274 at mRNA and protein levels in primary human monocytes.

<p>CD274 mRNA levels were determined by quantitative real-time PCR and are shown as fold changes in arbitrary units. (A) Representative western blot evaluating p65 protein levels after siRNA (negative control (NC) or p65-directed) nucleofection of primary human monocytes. LPS was added to cells after 24 h of transfection and incubated for 0, 12, 24 and 48 h. (B) Effect of LPS and p65 siRNA combined treatment on CD274 mRNA levels (n = 3, repeated three times, *P<0.05 vs. control with LPS treatment for 12 h). (C). Effect of LPS and p65 siRNA combined treatment on CD274 protein levels. CD274 protein levels were determined by flow cytometry. Dotted lines indicate background staining. Numbers in histograms indicate the geometric mean fluorescence of CD274-positive monocytes.</p

CD274 promoter analysis.

<p>(A). In silico analysis of an approximately 2.1 kb nucleotide sequence of the potential promoter and first exon of CD274. The first nucleotide of the 5′-RACE product is indicated by an arrow and assigned the nucleotide position +1. Putative NF-κB binding sites are boxed. (B). Partial sequencing result for the 5′RACE of the CD274 cDNA from a representative clone, red arrow represent the corresponding CD274 transcription start site.</p

CD274 promoter/reporter deletion analysis.

<p>Constructs were generated by progressively cloning 5′-truncated CD274 promoter fragments into the pGL3-basic luciferase vector. Negative numbers denote bp distances from the transcriptional start site (panel A). U937 cells were electroporation cotransfected with these reporter constructs and pRL-TK to control for transfection efficiency. Cells were either unstimulated (panel B) or stimulated with LPS (panel C) for 24 h.</p

NF-κB Plays a Key Role in Inducing <i>CD274</i> Expression in Human Monocytes after Lipopolysaccharide Treatment

<div><p>CD274, one of two co-stimulatory ligands for programmed death 1 and widely expressed in the mononuclear phagocyte system (MPS), may co-stimulate T cells and regulates inflammatory responses. However, changes in <i>CD274</i> gene expression and the underlying molecular mechanism are poorly understood during inflammatory responses. Therefore, delineation of the complex mechanisms regulating <i>CD274</i> expression is critical to understand this immunoregulatory system during inflammatory responses. The purpose of this study was to assess the molecular mechanisms regulating <i>CD274</i> expression in an <i>in vitro</i> monocyte model of inflammatory response. Firstly, <i>CD274</i> expression levels in human primary monocytes after lipopolysaccharide (LPS) treatment were observed and correlated with NF-κB activation. Secondly, based on the distribution of putative NF-κB binding sites, 5′ truncated human <i>CD274</i> promoter reporters were constructed, transfected into U937 cells and critical promoter regions for basal (nt −570 to +94) and LPS-induced (nt −1735 to −570) transcription were identified by dual luciferase assays. Finally, a key NF-κB binding site (nt −610 to −601) for LPS-inducible <i>CD274</i> transcriptional activity was characterized by point mutation analysis and chromatin immunoprecipitation analysis assays (ChIP). Thus, the present study establishes a molecular basis to understand the mechanisms governing <i>CD274</i> expression in certain infections and inflammatory disorders.</p></div

Chromatin immunoprecipitation (ChIP) assays of the CD274 promoter in primary human monocytes.

<p>Cells were treated with or without LPS for 1 h. ChIP assays were carried out using an anti-p65 antibody. IgG, anti-β-actin and anti-STAT1 antibodies were used as negative controls. Relative enrichment of each transcription factor-bound DNA was detected by qPCR using ChIP primers. All the results were normalized to input DNA.</p

Encapsulation of Laccase in Silica Colloidosomes for Catalysis in Organic Media

We report on the encapsulation of a laccase solution in silica colloidosomes of different shell structures and the study of catalytic performance of the encapsulated enzyme in toluene. The encapsulation is carried out by linking silica nanoparticles at the water/oil interface using hyperbranched polyethoxysiloxane (PEOS) in an aqueous-laccase-solution-in-toluene Pickering emulsion. By varying the weight ratio of silica particles to water (<i>R</i>_s/w), colloidosome shell can be adjusted from a particle monolayer up to a bilayer bound with a sandwiched nanoporous silica thin film formed from PEOS. The encapsulated laccase exhibits catalytic activity and reusability that are controlled by <i>R</i>_<i>s</i>/w. With the increase of <i>R</i>_s/w the reusability of the enzyme improves, meanwhile, its activity declines. This method allows fabricating enzyme microcapsules with tailored activity and controlled release properties

In vitro clonogenic survival and in vivo tumorigenic assays in MDA-MB453 tumor cells after knock-down of RecQL4 expression.

<p>(A) Western blot analysis of RecQL4 expression in parental, control shRNA (ShControl) and RecQL4 specific shRNA transduced MDA-MB453 cells (ShRecQL4-C5 & C8). RecQL4 expression was highly reduced in ShRecQL4-C5 and C8 stably selected by puromycin antibiotics. (B) Clonogenic survival assay was performed on ShRecQL4-C5 and C8 cells relative to parental and ShControl cells. Data presented at each time point were the mean value of eight cultures from two independent experiments. Bars indicate mean±SD. (C) Analysis of <i>in vivo</i> tumorigenicity of MDA-MB453 cells after RecQL4 silencing. Parental, ShControl and ShRecQL4-C5 & C8 cells were subcutaneously injected into seven immunosuppressed nude mice and tumor growth was monitored for 4 weeks. Tumor growth as a function of time is shown in the left panel. Images of tumors resected from mice are shown in the right panel. Note that 4 of 7 mice injected with either ShRecQL4-C5 or C8 cells did not show any tumor growth. Bars indicate mean±SD.</p

RecQL4 associates with survivin.

<p>(A) Endogenous survivin was immunoprecipitated with Flag-RecQL4 recognized by an anti-Flag antibody from cell extracts of 10⁶ U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were detected with antibody against survivin (Cell Signal). Five percent of the lysate was used for the loading control (Input) and the remaining 95% for co-immunoprecipitation. (B) Endogeneous RecQL4 was immunoprecipitated with Flag-survivin recognized by an anti-Flag antibody from cell extracts of 10⁶ U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were visualized by Western blot analysis with antibody against RecQL4 (Cell signal). (C) In the upper panel, schematic diagram of RecQL4 deletion constructs used for Co-IP studies is shown. In the lower panel, 293T cells were co-transfected with pRc-CMV2-survivin and one of the Flag-tagged truncated RecQL4 expressing vectors: pFlag-RecQL4-NT(−), pFlag-RecQL4-HD(−) or pFlag-RecQL4-CT(−). Survivin was immunoprecipitated with N-terminal (NT) deleted Flag-RecQL4 protein, not with helicase domain (HD) or C-terminal (CT) deleted Flag-RecQL4 protein.</p

RecQL4 protein or mRNA level in breast tumor cell lines and clinical breast cancer specimens.

<p>(A) Western blot detection of RecQL4 protein level in HMEC, MCF-10F and five breast tumor cell lines. β-Actin was used to verify equal loading of proteins. (B) Analysis of RecQL4 expression by quantitative real time PCR in normal breast tissues and breast cancer specimens with different pathological grades. TissueScan breast cancer tissue qPCR array was purchased from Origene. RecQL4 expression detected in normal breast tissues was considered as 1. The data represent mean ± SD from three independent experiments.</p