Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Antitumor and Angiostatic Activities of the Antimicrobial Peptide Dermaseptin B2

doi: 10.1371/journal.pone.0044351

Figure Lengend Snippet: Treatment of xenografted nude mice was started thirteen days after s.c. PC3 cell injection. Experimental group (n = 8) was treated six times per week p.t. with 100 µL Drs B2 (2.5 mg/kg body weight). Control group (n = 7) was treated six times per week p.t. with 100 µL of PBS. A ) The effect of Drs B2 on tumor size versus time of treatment. After 47 days of treatment the mice were sacrificed, body weight was observed; tumors were isolated, B ) weighted and stored at −80°C. C ) PC3 tumor proliferation was evaluated by Ki67 staining of frozen tissue sections. Proliferation was quantified by image J software analysis of Ki67 positive stained cells on the whole tumor section. The data are mean areas ± SD obtained from 6 control mice and 4 mice treated with Drs B2. Scale bar, 50 µm. * p <0.05 versus control (PBS). D ) Tumor vessel formation was observed with anti CD34 antibodies. Angiogenesis was quantified by image analysis of CD34 positive stained endothelial cells. The data are mean areas ± SD obtained from 6 control mice and 4 mice treated with Drs B2. Scale bar, 50 µm * p <0.05 versus control (PBS).

Article Snippet: Mouse monoclonal anti-CD34 was from Hycult Biotech (The Netherlands), secondary antibody, rabbit anti-rat IgG was obtained from Southern Biotech (Clinisciences; France).

Techniques: Injection, Isolation, Staining, Software

Journal: Nature

Article Title: Inhibition of LTβR-signalling activates Wnt-induced regeneration in lung

doi: 10.1038/s41586-020-2882-8

Figure Lengend Snippet: a-b, Representative images of immunohistochemical analysis for RelA ( a ) and RelB ( b ) (brown signal, indicated by arrows, haematoxylin counter stained, scale bar 50μm, zoomed area 25μm) in lung core biopsy sections from healthy (n=3) and COPD patients (n=4), with the quantification of RelA and RelB positive alveolar epithelial nuclei shown as mean ± SD. c-e , Gene set enrichment analysis (GSEA) of the LTβR signalling, NF-κB signalling (gene lists from IPA software, Qiagen), TNFR-mediated signalling (GO:0033209), positive regulation of I-kappaB kinase NF-kappaB signalling (GO:0043123) and NIK NF-kappaB signalling (GO:0038061) pathways in publically available array data from lung tissue (GSE47460-GPL14550) of healthy (n=91) v COPD patients (n=145) ( c ), from lung tissue (GSE37768) of healthy (n=9) v COPD patients (n=18) ( d ) and from peripheral blood mononuclear cells (GSE56768) of healthy (n=5) v COPD patients (n=49) ( e ). f , mRNA expression levels of Lta , Ltbr , Tnfsf14 ( Light ), Tnf , Ccl2 and Cxcl13 determined by qPCR in whole lung from B6 mice exposed to filtered air (FA, n=6) or cigarette smoke (CS, n=8) for 6m, individual mice shown. g , GSEA of the pathways described in (c-e) in the publically available array data (GSE52509) of lungs from our mice exposed to filtere air (FA, n=3) and cigarette smoke (CS, n=6) for 4 and 6m. h , Western blot analysis for RelB, p100 and p52 in total lung homogenate from the mice described in (f). Quantification relative to vinculin of individual mice shown (n=3). For gel source data see . i , Schematic representation of the LTβR-Ig treatment protocol. j , Representative low and high magnification overlay images of Multiplex immunofluorescence staining to identify CD4 (Red), CD8 (Green), B220 (Turquoise) and DAPI (blue) counterstained lung sections (Scale bars 100μm, n=4) from B6 mice exposed to CS for 6m, plus LTβR-Ig fusion protein or control Ig (80 μg i.p., weekly) therapeutically from 4 to 6m, and analysed at 6m. k , mRNA expression levels of Cxcl13 and Ccl19 determined by qPCR in whole lung from B6 mice exposed to FA or CS for 4 and 6m, plus LTβR-Ig fusion or control Ig (80 μg i.p., weekly) prophylactically from 2 to 4m and analysed at 4m, and therapeutically from 4 to 6m and analysed at 6m (n=4 mice/group, repeated twice, pooled data shown). P values indicated, Mann-Whitney one-sided test (a-b), unpaired two-tailed Student’s t test (f, h), one-way ANOVA multiple comparisons Bonferroni test (k).

Article Snippet: 24 h later, cells were stimulated for 6 and 24 h with an agonistic antibody to LTβR [2 μg/ml] (5G11, Cat. No. HM1079, Hycult Biotech), recombinant murine TNF-α [1 ng/ml] (Cat. No. 315–01A, PeproTech) or a combination thereof.

Techniques: Immunohistochemical staining, Staining, Software, Expressing, Western Blot, Multiplex Assay, Immunofluorescence, MANN-WHITNEY, Two Tailed Test