Journal: Journal of Biological Chemistry

Article Title: Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

doi: 10.1074/jbc.m611125200

Figure Lengend Snippet: FIGURE 7. The DNA binding activity of Ku at RAG bound signal ends. A, a signal end complex is defined here as RAG1, RAG2, and HMG1 bound to two- oligonucleotide DNA duplexes, one containing the 12-RS and the other con- taining the 23-RS. A streptavidin-biotin complex was used to block the ends of the DNA duplexes distal to the site of cleavage. B, approximately 10 ng/l of purified RAG1 and RAG2 and 425 nM HMG1 were incubated with 0.4 nM radiolabeled23-RS-containingand10nM12-RS-containingDNAfragmentsat 37 °C for 10 min. 5 M streptavidin was added at 25 °C for 5 min prior to addition of 25 nM Ku. Antibody supershifts required an additional 10-min room temperature incubation step with either 0.2 g of the monoclonal anti- body to Ku or 1 l of a polyclonal antisera recognizing the maltose binding domain fused to recombinant RAG proteins. The inferred composition of each of the generated species is indicated to the side of the panel: species I, 23-RS; species II, HMG1-bound 23-RS; species III, RAGs and HMG1 bound to the 23-RS; species IV, SEC; species V, streptavidin-blocked SEC (upper arrow indi- cates -MBP supershift); species VI, Ku bound to incompletely formed SEC (upper arrow indicates -Ku supershift).

Article Snippet: The relative amounts of Ku and histoneH3 in each of the excised complexes were determined by semi- quantitative Western analysis probing with a polyclonal rabbit antibody raised against native, recombinant human Ku and a polyclonal antibody against histone H3 (Ab1791; Abcam) using fluorescent detection and a Typhoon imager (GE Healthcare).

Techniques: Binding Assay, Activity Assay, Blocking Assay, Purification, Incubation, Recombinant, Generated

Journal: Nature Communications

Article Title: The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus

doi: 10.1038/s41467-019-14191-1

Figure Lengend Snippet: a , b A549 epithelial cells or macrophages were infected with the wild-type (WT), the nctA null mutant ( ∆nctA ) or the nctA reconstituted isolate ( nctA rec ) for 24 h. Cytotoxicity of each mutant was evaluated by measuring the release of lactate dehydrogenase (LDH) activity into the culture medium. The data represent five different infection challenges with triplicate LDH activity measurements. Data are shown in fold change of LDH activity relative to the wild-type-infected cells. The error bars mean the standard error of the mean (SEM), and P- values were calculated by Kruskal–Wallis test with Dunn’s correction: * P < 0.0180; ** P < 0.0056 (for a , A549 cells). ** P < 0.0032; *** P < 0.0007 (for b , macrophages). c – f Granulocyte macrophage colony-stimulating factor-induced bone marrow-derived dendritic cells (gm-csf BMDCs) were infected with the A. fumigatus strains with the multiplicity of infection (MOI) = 5:1. Proinflammatory cytokines were quantified by ELISA. Data represent three biological replicates ( c – e ) or five biological replicates ( f ) with ± SEM. P- values were calculated by ANOVA with Tukey’s correction: *** P < 0.002; **** P < 0.0001. g , h Activation of dendritic cells measured by CD40 and CD80 markers by flow cytometry. Data represent three biological replicates with ±SEM. P -values were calculated by ANOVA: *** P < 0.0002; **** P < 0.0001 (for g ). *** P < 0.0004; **** P < 0.0001 (for h ). Source data are provided as a Source Data file.

Article Snippet: The concentration of IL-8 and IL-6 were determined in A549 epithelial cells co-cultured with A. fumigatus strains by using the Human IL-8/CXCL8 and IL-6 DuoSet ELISA according to the manufacturer’s instructions (R&D systems).

Techniques: Infection, Mutagenesis, Activity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Flow Cytometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: siRNA Library Screening Identifies a Druggable Immune-Signature Driving Esophageal Adenocarcinoma Cell Growth

doi: 10.1016/j.jcmgh.2018.01.012

Figure Lengend Snippet: LIF hierarchically regulates STAT3 phosphorylation, IL6 expression, and cell proliferation in EAC cells. SiRNA-mediated silencing of LIF expression results in reduced LIF mRNA ( A ) expression, ( B ) secretion, and ( C ) a recombinant-recoverable reduction in cell proliferation in CP-D, SKGT4, and OE-33 EAC cell lines. Treatment of LIF silenced cells with recombinant LIF protein (rLIF) results in ( D ) recovery of LIF mRNA in CP-D, SKGT4, and OE33 EAC cells and ( E ) protein expression levels in SKGT4 EAC cells. ( F ) STAT3-Y705 phosphorylation levels in LIF-silenced SKGT4 EAC cells are rescued by treatment with recombinant LIF protein as determined by Western blot. ( G ) Silencing LIF expression in SKGT4 EAC cells results in decreased IL6 mRNA expression that is recovered upon exposure to rLIF protein. ( H ) Neither silencing IL6 expression by siRNA-mediated silencing nor treatment with anti-IL6 antibody affects LIF mRNA levels or SKGT4 EAC cell growth. ( I ) Silencing LIF expression results in decreased expression of C1QA mRNA but not TREM2 in SKGT4 EAC cells. ∗ P < .05, ** P < .01, *** P < .001, and **** P < .0001 in either Student t test (proliferation) or Mann–Whitney testing (mRNA expression). siNT, nontargeting siRNA pool; siLIF, LIF-targeted siRNA pool; rLIF, recombinant LIF protein; rIL6, recombinant IL6 protein.

Article Snippet: Treatments with recombinant leukemia inhibitory factor (LIF) (30 ng/mL, ab57665; Abcam), IL6 (30 ng/mL; R&D Systems, Minneapolis, MN), native C1q (75 μg/mL; Sigma, Oakville, Ontario), and IL6 blocking antibody (AB206NA) experiments were performed in either 6- or 96-well cell culture plates for protein and viability experiments, respectively.

Techniques: Phospho-proteomics, Expressing, Recombinant, Western Blot, MANN-WHITNEY