Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: The 9 bp sequence in the 5′-untranslated region (UTR) of Far2 binds NKX3-2. A: electrophoretic mobility shift assay (EMSA) performed with nuclear extract from mouse mesangial cells incubated with biotin-labeled oligonucleotide probes corresponding to the Far2 5′-UTR sequences with (ins-probe) and without (del-probe) the 9 bp sequence (lanes 2 and 4, respectively). B: EMSA performed with purified NKX3-2 protein (lane 2). A supershift assay was performed with an antibody against NKX3-2 (lane 3).

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Sequencing, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Purification

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: Far2 is an enzyme involved in de novo platelet-activating factor (PAF) production. A: PAF concentration measured in both culture media and cell lysates between Far2-overexpressing (gray, n = 3) and wild-type (black, n = 3) mesangial cells. Labeled fatty alcohol (hexadecanol) (B) and PAF (C) were detected through mass spectrometry after a mixture of 13C- and 12C-palmitate was added to Far2-overexpressing cells. D: proposed pathway highlighting the role of FAR2 in de novo synthesis of PAF.

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Concentration Assay, Labeling, Mass Spectrometry

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: Generation and characterization of B6N(Cg)-Far2tm2a(KOMP)Wtsi/2J. A: a simplified scheme showing the wild-type Far2 allele and knock-in allele Far2tm2a(KOMP)Wtsi. The lacZ and neomycin expression cassette was inserted between exon 4 and exon 5 and is flanked by FRT (Flippase Recognition Target) sites. Exons 5–8 are flanked by loxP sites. B: quantitative PCR for Far2 on brain and eyelid of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) knockout mice (n = 3/genotype) levels were calculated as relative fold change (RFC) compared with wild type. *P < 0.01 compared with +/+; #P < 0.01 compared with +/−. C: quantification of mesangial matrix expansion (MME) in knockout mice vs. wild-type mice at 12 and 18 mo of age relative to 6 mo measurements. MME is reported as percentage of the glomerular area occupied by matrix for 50 glomeruli per mouse and 10 mice per group. P = 1.27 × 10−6. D: glomerular filtration rate (GFR) data in knockout mice (gray) vs. wild-type mice (black) at 6, 12, and 18 mo of age. n = 10 per group. *P = 0.0118.

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Knock-In, Expressing, Real-time Polymerase Chain Reaction, Knock-Out, Filtration

Journal: Respiratory Research

Article Title: Aggregatibacter is inversely associated with inflammatory mediators in sputa of patients with chronic airway diseases and reduces inflammation in vitro

doi: 10.1186/s12931-024-02983-z

Figure Lengend Snippet: Determination of the minimal effective MOI (mMOI) of a ) A. actinomycetemcomitans and b ) A. aphrophilus based on NF-κB pathway activation and determination of the mMOI of c ) A. actinomycetemcomitans and d ) A. aphrophilus based on IL-8 production in 3-D lung epithelial cells. On the vertical axis % NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells is shown, on the horizontal axis the tested MOI is depicted. The mMOI was defined as the lowest MOI with anti-inflammatory activity (i.e. < 50% NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells as visualized by a dotted line, p < 0,05). e ) Western blot analysis of proteins (i.e. IκBα, p65 and phosphorylated (p)-IκBα) produced by 3-D A549 cells stimulated with LPS in the presence or absence of A. actinomycetemcomitans (targeted MOI 50:1) for 4 h (representative replicate is shown, image contains cropped blot – the full original blot is shown in Supplementary file_Western blot image). f ) Band intensity (normalised to β-actin) of Western blot at 4 h of 3-D A549 cells stimulated with LPS with/without A. actinomycetemcomitans . Results are expressed as a percentage of the positive control (i.e. LPS). NC: negative control (untreated 3-D A549 cells in serum-free GTSF-2 medium), A.ac. A. actinomycetemcomitans . Data represent mean ± SEM, * p < 0.05, n ≥ 3

Article Snippet: A previously developed organotypic 3-D lung cell culture model of the A549 alveolar epithelial cell line (ATCC CCL185) or the NF-κB–luciferase-transfected A549 recombinant stable cell line (BPS Bioscience, San Diego, CA, US) was used that exhibits in vivo-like phenotypic and functional properties of alveolar epithelial cells, including barrier function (localized expression of tight junctional markers), apical and basolateral polarity, and responds to infection in ways that are relevant to the infection process in vivo, including cytokine secretion [ , – ].

Techniques: Activation Assay, Activity Assay, Western Blot, Produced, Positive Control, Negative Control

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: Colocalisation of the ovine OX40L and CD70 ligands with their cognate receptors. ( A ) Immunolocalisation of Oa OX40 and Oa CD27 in transfected HEK293 cells detected by anti-V5 tag antibody and counterstained with DAPI. Cells expressing ( B ) Oa OX40 or ( D ) Oa CD27, were incubated with 10 μg of ( B ) purified r Oa OX40L or ( D ) purified r Oa CD70 for 15 min before fixation and immunofluorescence. Oa OX40 and Oa CD27 expression was detected using anti-V5 tag antibodies (red); r Oa OX40L and r Oa CD70 presence were detected with an anti-ovine Fc antibody (green). ( B , D ) Insets show detail of colocalisation in each case. Merge and orthogonal projection were obtained with ImageJ software. Scale bar = 10 μm, indent scale bar = 4 μm. In panel ( C ) and ( E ), an enlarged image of corresponding insets in Merge and fluorescence intensity profiles for the indicated white lines on the images are shown. ( F ) ImageJ Image calculator function was used to evaluate colocalisation of ligand signal with its receptor. Representative images of colocalisation analysis in a Z-plane (presented as 16LUT signal) are shown. The ImageJ calculator tool was used for pixel colocalisation for the fluorescence channels of ligands (r Oa OX40L or r Oa CD70) and their respective receptors (r Oa OX40 or r Oa CD27). ( G ) The percentage of recombinant ligand signals colocalising with their receptors in ( F ) was analysed in 25–40 cells for each condition. Mean ± SD are indicated for each condition.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Expressing, Incubation, Purification, Immunofluorescence, Software, Fluorescence, Recombinant

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: The secreted r Oa OX40L and r Oa CD70 expressed from recombinant adenovirus-infected cells colocalise with their cognate ovine receptors. Cells expressing ( A ) Oa OX40 or (C) Oa CD27, were incubated with conditioned media from cells infected with ( A , B ) Ad5- Oa OX40L (r Oa OX40L) or ( C , D ) Ad5- Oa CD70 (r Oa CD70) for 15 min before fixation and immunofluorescence. r Oa OX40L and r Oa CD70 were detected with an anti-Fc antibody (green); Oa OX40 and Oa CD27 expression was detected using anti-V5 tag antibodies (red). ( B , D ) Insets show detail of colocalisation for each case. Merge and orthogonal projections were obtained with ImageJ software. Scale bar = 10 μm, indent scale bar = 4 μm. In panel ( B ) and ( D ), an enlarged image of corresponding insets in Merge and fluorescence intensity profiles for the indicated white lines on the images are shown. ( E ) Representative images of colocalisation analysis in a Z-plane (presented as 16LUT signal) are shown. The ImageJ calculator tool was used for pixel colocalisation for the fluorescence channels of ligands (r Oa OX40L or r Oa CD70) and their respective receptors (r Oa OX40 or r Oa CD27). ( F ) The percentage of ( E ) adenovirus-produced ligands signal colocalising with their receptors was analysed in 25–40 cells for each condition. Mean ± SD are indicated for each condition.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Recombinant, Infection, Expressing, Incubation, Immunofluorescence, Software, Fluorescence, Produced

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: The r Oa OX40L and r Oa CD70 specifically elicit signalling through their cognate receptors Oa OX40 and Oa CD27. HEK293-pr(IFNB)-GFP cells were transfected with, ( A – C ) Oa OX40-V5 and ( D – F ) Oa CD27-V5. At 24 h post transfection, the cells were stimulated with 10 μg of purified ( A ) rOa OX40L or ( D ) rOa CD70 proteins, or equivalent amounts of conditioned media from Ad5- Oa OX40L ( B ) rOa OX40L, ( E ) Ad5- Oa CD70, or ( C , F ) Ad5-DsRed infected Vero cells proteins in conditioned media from Ad5- Oa OX40L, Ad5- Oa CD70, or Ad5-DsRed infections. At 16 h post stimulation, the cells were fixed and an immunofluorescence using anti-V5 (red) to detect the transfected receptors was performed. GFP (green) activity was detected by fluorescence microscopy and DAPI was used to counterstain cells to nuclei (blue). Bars = 20 µm.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Purification, Infection, Immunofluorescence, Activity Assay, Fluorescence, Microscopy

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: Dose dependent induction of signalling by r Oa OX40L and r Oa CD70 ligands. HEK-293/prIFNB-GFP cells were transfected with plasmids to express the receptors Oa OX40 or Oa CD27 and stimulated at 24 h post transfection with three equivalent and increasing doses (1×, 2×, and 4×) of conditioned media from Ad5- Oa OX40L (r Oa OX40L) or Ad5- Oa CD70 (r Oa CD70) infected Vero cells. As a positive control of signalling induction, untransfected cells were infected with Sendai virus (SeV) at a multiplicity of infection (moi) of 1 pfu/cell for 16 h. GFP expressing cells were counted on an immunofluorescence microscope on 12 randomly chosen fields and % of GFP expressing cells for each field are plotted. Mean ± SD for each condition are indicated with bars.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Infection, Positive Control, Virus, Expressing, Immunofluorescence, Microscopy

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: CD27 expression on ovine PBMC populations. Ovine PBMC, derived from n = 15 animals were costained for CD27 and CD4, CD8, CD335, or B cell markers. Gates were set using the corresponding isotype controls. Isotype control staining and representative dot plots for CD27 staining and CD4; CD8; CD335 or B cell marker are shown. Bar charts show the mean (±SD) percentage of CD27 + and CD27 - cells in the CD4/CD8/CD335/B cell marker gates (upper quadrants) in donor sheep PBMC.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Expressing, Derivative Assay, Control, Staining, Marker