Journal: International Journal of Molecular Sciences

Article Title: Gap Junctions between Endothelial Cells Are Disrupted by Circulating Extracellular Vesicles from Sickle Cell Patients with Acute Chest Syndrome

doi: 10.3390/ijms21238884

Figure Lengend Snippet: Effect of ACS plasma-derived EVs on the extent of gap junction-mediated intercellular communication. ( A ) Representative photomicrographs of intercellular transfer of Neurobiotin after its injection into a single cell (indicated by arrows) within a monolayer of endothelial cells, treated for 48 h with EVs, isolated at baseline and during an episode of ACS. The Neurobiotin tracer was detected after staining the cells with streptavidin-Cy3 conjugate (yellow). Nuclei were stained with DAPI (blue) to show the locations of all cells. ( B ) Graph shows the quantitation of Neurobiotin transfer experiments (numbers of tracer-containing neighbors) ( n = 6–9 injections for each condition). The ACS EVs caused decreased Neurobiotin transfer to neighboring cells (decreased intercellular communication) (★ = paired t -test comparing ACS and baseline within SCD subjects, p < 0.05).

Article Snippet: Individual cells within clusters were impaled and injected with 5% Lucifer yellow (charge = −2, MW = 456; Thermo Fisher Scientific Inc.) and 5% Neurobiotin (charge = +1, MW = 322.8; Vector Laboratories, Burlingame, CA dissolved in water) for 1 min using a picospritzer (model PLI-188, Nikon Instruments, Melville, NY, USA).

Techniques: Derivative Assay, Injection, Isolation, Staining, Quantitation Assay

Journal: Infection and Immunity

Article Title: Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

doi: 10.1128/IAI.02060-14

Figure Lengend Snippet: Phospholipase D and diacylglycerol lipase contribute arachidonic acid for PGE2 biosynthesis. (a) RAW 264.7 macrophages labeled with [3H]arachidonic acid were treated with 10 μM VU0155056 1 hour prior to inoculation with F. tularensis LVS. Twenty hours after inoculation, radioactivity in the supernatant and cell fraction was measured by liquid scintillation and expressed as percent release (n = 3). (b) BMDMs or RAW 264.7 macrophages were pretreated with inhibitors and then inoculated with F. tularensis LVS at an MOI of 200:1. Three hours after inoculation, 5 μM arachidonic acid was added to culture medium. PGE2 in the supernatant was quantified by enzyme-linked immunosorbent assay 24 hours after inoculation (n = 4). Asterisks denote a statistical difference compared to the LVS-infected group (a) or the corresponding AA-treated group (b) (P ≤ 0.05). For clarity, the data from some experiments were presented in multiple figures. However, these experiments were run simultaneously and as such utilize the same controls (uninfected, infected, and COX-2 inhibitor treated). This includes Fig. 2c and ​anddd and panels a and b of this figure. Graphs are means of pooled experiments ± SEMs.

Article Snippet: The inhibitors used in these studies were pyrrophenone (for cPLA 2 ), CAY10590 (for sPLA 2 ), Edelfosine (for phosphatidylinositol [PI]-PLC), JZL184 (for MAGL), and indomethacin (for COX-2) (all purchased from Cayman Chemical); propranolol (for PAP), RHC80267 (for DAGL), MJ33 (for iPLA 2 ), {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 (for PLC), and cytochalasin D (CD) (for actin polymerization) (all purchased from Sigma-Aldrich); VU0155056 (for PLD1/2) (purchased from Avanti Polar Lipids, Inc.); and D609 (for phosphatidylcholine [PC]-PLC) (purchased from Tocris Bioscience).

Techniques: Labeling, Radioactivity, Enzyme-linked Immunosorbent Assay, Infection

Journal: Infection and Immunity

Article Title: Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

doi: 10.1128/IAI.02060-14

Figure Lengend Snippet: Inhibitors used in this study a

Article Snippet: The inhibitors used in these studies were pyrrophenone (for cPLA 2 ), CAY10590 (for sPLA 2 ), Edelfosine (for phosphatidylinositol [PI]-PLC), JZL184 (for MAGL), and indomethacin (for COX-2) (all purchased from Cayman Chemical); propranolol (for PAP), RHC80267 (for DAGL), MJ33 (for iPLA 2 ), {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 (for PLC), and cytochalasin D (CD) (for actin polymerization) (all purchased from Sigma-Aldrich); VU0155056 (for PLD1/2) (purchased from Avanti Polar Lipids, Inc.); and D609 (for phosphatidylcholine [PC]-PLC) (purchased from Tocris Bioscience).

Techniques:

Journal: Infection and Immunity

Article Title: Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

doi: 10.1128/IAI.02060-14

Figure Lengend Snippet: Inhibition of macrophage cPLA2 (pyrrophenone), PAP (propranolol), DAGL (RHC80267), or PLD (VU0155056) reduced F. tularensis-induced prostaglandin E2 biosynthesis by infected macrophages. (a) F. tularensis LVS was inoculated in broth medium and left untreated or incubated with inhibitors/vehicle and allowed to grow over 12 hours. Bacterial growth was measured as a function of absorbance at 600 nm. Curves represent average growth from 3 independent experiments. (b) BMDMs or RAW 264.7 macrophages were pretreated with inhibitors and then inoculated with F. tularensis LVS at an MOI of 200:1; PGE2 in the supernatant was quantified by enzyme-linked immunosorbent assay 24 hours after inoculation. Data represent means ± SEMs. n = 9 (BMDMs); n = 17 (RAW cells). (c) LIVE/DEAD Fixable Yellow stain analysis was performed to verify that inhibitors were not causing cell death or increased cell death during F. tularensis infection. BMDMs or RAW 264.7 macrophages were incubated with vehicle or inhibitor for 20 hours at 37°C (n = 6). Asterisks denote a statistical difference compared to the untreated infected group (P ≤ 0.05). Graphs are means of pooled experiments ± SEMs.

Article Snippet: The inhibitors used in these studies were pyrrophenone (for cPLA 2 ), CAY10590 (for sPLA 2 ), Edelfosine (for phosphatidylinositol [PI]-PLC), JZL184 (for MAGL), and indomethacin (for COX-2) (all purchased from Cayman Chemical); propranolol (for PAP), RHC80267 (for DAGL), MJ33 (for iPLA 2 ), {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 (for PLC), and cytochalasin D (CD) (for actin polymerization) (all purchased from Sigma-Aldrich); VU0155056 (for PLD1/2) (purchased from Avanti Polar Lipids, Inc.); and D609 (for phosphatidylcholine [PC]-PLC) (purchased from Tocris Bioscience).

Techniques: Inhibition, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Staining