assay kit  (Echelon Biosciences)

Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Immunofluorescence was used to detect autotaxin (ATX) expression in visceral and parietal tissues from Wt1 cre/+ ; R26R eYFP/+ reporter mice. Cells that express Wt1, a mesothelial marker, will express enhanced yellow fluorescent protein (eYFP) that can be detected with a green fluorescent protein (GFP) antibody (A, D, G). Robust autotaxin expression is evident in visceral mesothelia (A–F). In contrast, the parietal mesothelium that lines the body wall expresses the mesothelial marker (G), but is devoid of autotaxin expression (H). Nuclei are marked with DAPI. Quantitative real-time RT-PCR was used to determine autotaxin transcript levels in visceral (omental) mesothelium, parietal mesothelium, heart, and liver (J). The autotaxin expression in visceral cells was set to 1 and fold changes were calculated for each subsequent tissue type. The asterisk represents a statistically significant difference when compared to the expression level in visceral cells (p<0.05, n = 4). Error bars were calculated using standard error of the mean.

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Immunofluorescence, Expressing, Marker, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Conditioned media was collected from cultured visceral mesothelia, parietal mesothelia, peritoneal mesothelioma (MSTO), and pleural mesothelioma (ROB) cells. Some cultures were treated with the autotaxin inhibitor S32826. Media was assayed for LPA accumulation as a measure of autotaxin activity over a 2 hour period. Visceral mesothelia had significantly higher levels of autotaxin activity compared to parietal cells (A). Furthermore, MSTO and ROB cells had even higher levels of autotaxin activity compared to normal mesothelial cells (B). Normal and pathological cells were responsive to S32826 and displayed decreased levels of autotaxin activity (A–B).

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Cell Culture, Activity Assay

Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Visceral mesothelial, parietal mesothelial, pleural mesothelioma (MSTO), and peritoneal mesothelioma (ROB) cells were cultured on transwell filters with and without addition of autotaxin (ATX), the autotaxin inhibitor S32826, Lpar1 inhibitor 1 (2440), or Lpar1 inhibitor 2 (8437). Of all the cells studied, the mesothelioma cells were the most invasive (C, D). Visceral cells (A) invaded more readily than parietal (B), but addition of ATX to parietal cells (F) promoted invasion to visceral-like levels. Treatment with S32826 reduced cell invasion in all groups (I-L). Furthermore, both of the Lpar1 inhibitors were efficient at decreasing cell invasion in mesothelial (M, Q) and mesothelioma cells (O, P, S, T). The percent of cell invasion is quantified in panel U. The percent of untreated visceral cells that migrated was set to 100. Asterisks represent statistically significant differences compared to untreated values in each group (p<0.05, n = 4). Error bars were calculated using standard error of the mean.

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Visceral mesothelial, parietal mesothelial, pleural mesothelioma (MSTO), and peritoneal mesothelioma (ROB) cells were plated on fibronectin, cultured with or without the addition of autotaxin, the autotaxin inhibitor S32826, Lpar1 inhibitor 1 (2440), or Lpar1 inhibitor 2 (8437), and subjected to time-lapse imaging in order to visualize their ability to migrate. Panels A-H are compilations of static images taken at 20 minute intervals. False coloration indicates a cell's location at each interval: red 0 min; green 20 min; yellow 40 min; blue 60 min; purple 80 min.

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Cell Culture, Imaging

Journal: Molecular Cancer

Article Title: Autotaxin expression and its connection with the TNF-alpha-NF-κB axis in human hepatocellular carcinoma

doi: 10.1186/1476-4598-9-71

Figure Lengend Snippet: Up-regulation of ATX induced by TNF-α is associated with increased lysophospholipase D (lyso-PLD) activity by conversion of LPC into LPA in Hep3B and Huh7 cells . Serum starved Hep3B or Huh7 cells were treated with TNF-α (10 ng/ml) or vehicle (0.1%BSA/PBS) for 20 hours. A . Conditioned media (CM) or control media (DMEM or EMEM) were concentrated (40-fold) and assayed for ATX activity using the FS-3 compound. The results are shown as the average of relative fluorescence activity ± SD from three experiments. B . CM were incubated with 15 μM LPC (18:1) for 3 hours at 37°C. Lipids were analyzed by liquid chromatography-mass spectrometry (LC-MS). Results are level of LPA (18:1) from three experiments and presented as mean ± SD. *, P < 0.05.

Article Snippet: ATX activity assay reagents were from Echelon Biosciences, Inc. (Salt lake City, UT, USA).

Techniques: Activity Assay, Fluorescence, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS ONE

Article Title: A Metabolically-Stabilized Phosphonate Analog of Lysophosphatidic Acid Attenuates Collagen-Induced Arthritis

doi: 10.1371/journal.pone.0070941

Figure Lengend Snippet: A–C. Steady-state hydrolysis of LPC (16:0, 18:0, 18:1 respectively) by recombinant ATX as measured with the TOOS activity assay. D–E. ATX activity inhibition dose-response curves in the presence of various BrP-LPA concentrations as measured with the TOOS assay using as substrates 50 μΜ of 16:0 (D) and 18:0 LPC (E). F–G . Cornish-Bowden (F) and Lineweaver-Burk (G) plots show that BrP-LPA is a competitive ATX inhibitor. H. The kinetic parameters (k m , V max ) obtained by the incubation of ATX at various concentrations of 16:0 LPC (100–400 μΜ) in the absence or presence of BrPLPA (0–10 μΜ). All presented values are the means (±std) of two independent experiments.

Article Snippet: Assays were conducted in a final volume of 100 μl in the presence of 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 and 50 mM Tris, pH 8.0 with the use of 1 µM FS-3, 2 nM ATX from Echelon and increasing concentrations of BrP-LPA.

Techniques: Recombinant, Activity Assay, Inhibition, Incubation

Journal: PLoS ONE

Article Title: A Metabolically-Stabilized Phosphonate Analog of Lysophosphatidic Acid Attenuates Collagen-Induced Arthritis

doi: 10.1371/journal.pone.0070941

Figure Lengend Snippet: A. Effect of BrP-LPA on plasma ATX/lysoPLD hydrolysis of exogenous added 50 μΜ LPC (14:0, 16:0, 18:0). B. Correlation of recovered BrP-LPA in whole blood with the added BrP-LPA. C. Inhibition of endogenous ATX/lysoPLD activity in whole blood ex vivo after the addition of increasing BrP-LPA concentrations (0–10 μΜ). ATX activity was measured in the presence of 1 mM LPC with the TOOS assay. D. LPC levels and E. LPA levels measured in whole blood ex vivo in the absence/presence of different BrP-LPA concentrations (0.03–10 μΜ). F. Per cent residual levels of the indicated LPA species in the presence of increasing amounts of BrP-LPA. Solid lines, best fits of averaged data points. * indicates significant (p<0.05), *** indicates significant (p<0.001) decrease relative to control group.

Article Snippet: Assays were conducted in a final volume of 100 μl in the presence of 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 and 50 mM Tris, pH 8.0 with the use of 1 µM FS-3, 2 nM ATX from Echelon and increasing concentrations of BrP-LPA.

Techniques: Inhibition, Activity Assay, Ex Vivo

Journal: PLoS ONE

Article Title: A Metabolically-Stabilized Phosphonate Analog of Lysophosphatidic Acid Attenuates Collagen-Induced Arthritis

doi: 10.1371/journal.pone.0070941

Figure Lengend Snippet: A. Plasma BrP-LPA pharmacokinetic profile following i.p administration of 10 mg/Kg BrP-LPA in female mice. B. Plasma per cent (%) residual ATX activity measured in the presence of 1 mM LPC with the TOOS assay and C . Plasma % residual total LPA levels at different time points after i.p. administration of BrP-LPA. D. Plasma concentration of different LPA and E. LPC species at different time points following BrP-LPA i.p administration. The time point 0 refers to the vehicle control. The presented values are the means (±std) of two independent experiments. *(p<0.05) and **(p<0.01) indicate a significant decrease relative to control group.

Article Snippet: Assays were conducted in a final volume of 100 μl in the presence of 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 and 50 mM Tris, pH 8.0 with the use of 1 µM FS-3, 2 nM ATX from Echelon and increasing concentrations of BrP-LPA.

Techniques: Activity Assay, Concentration Assay