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: Science Advances

Article Title: Identification of the NRF2 transcriptional network as a therapeutic target for trigeminal neuropathic pain

doi: 10.1126/sciadv.abo5633

Figure Lengend Snippet: Key resources table. N/A, not available.

Article Snippet: NRF2/ARE Luciferase Reporter HEK-293 Stable Cell Line , Signosis Inc. , Cat# SL-0042-NP.

Techniques: Recombinant, Modification, Protease Inhibitor, Transfection, Peroxidation Assay, Enzyme-linked Immunosorbent Assay, Luciferase, RNA Sequencing, Gene Expression, Stable Transfection, Sequencing, Cloning, Construct, Plasmid Preparation, Software