ethylene glycol bis beta aminoethyl ether n n n n tetraacetic acid tetrasodium salt  (Millipore)

Journal: Cell Death & Disease

Article Title: Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization

doi: 10.1038/cddis.2015.96

Figure Lengend Snippet: ( a ) Cell-free tubulin polymerization assay in vitro . Purified tubulin was used to test the ability of BPT to inhibit tubulin polymerization in vitro . The assay measures the increase in optical density as a result of tubulin assembly or polymerization. Nocodazole and paclitaxel were used in the assay as a known inhibitor and enhancer of tubulin polymerization. BPT was tested at four different concentrations that show inhibition of cell growth in vitro . The change in V max value was used as an indicator of tubulin/ligand interactions. The polymerization curves indicate 0.5 , 1, 2.5 and 5 μ M of BPT reduced the V max value from 19 mOD/min (control) to 12.5, 9.2, 3 and 0.5 mOD/min, respectively, in a dose-dependent manner. The curves shown represent the average of three independent experiments. ( b ) Inhibition of tubulin polymerization and enhancement of tubulin depolymerization in live cells. The tubulin polymerization assay was performed in A549 (whole cells) after 30 min compound treatment at the concentrations indicated in the figure. Supernatant and pellet represent unassembled and assembled tubulin, respectively. Tubulin polymerization is detectable by the increase of tubulin in pellet and its disappearance from supernatant. The western blots show dose-dependent inhibition of tubulin polymerization after the simultaneous treatment of paclitaxel and BPT that resulted in the accumulation of unassembled tubulin in supernatant. BPT also acts as an enhancer for tubulin depolymerization in a dose-dependent manner when paclitaxel-stabilized tubulin was subjected to BPT treatment for 30 min

Article Snippet: The plates were further incubated for 30 min, the cell monolayers were washed two times with sterile PBS at room temperature and then 100 μl tubulin extraction buffer (1 mM MgCl2 , 2 mM EGTA, 0.5% NP40 and 20 mM Tris-HCl (pH 6.8)) supplemented with 2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aldrich; cat. no. P8340) was added per well.

Techniques: Polymerization Assay, In Vitro, Purification, Inhibition, Western Blot

Journal: PLoS ONE

Article Title: Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death

doi: 10.1371/journal.pone.0075652

Figure Lengend Snippet: Macrophage death caused by Leptospira -induced [Ca 2+ ]i elevation. (A). High [Ca 2+ ]i-related apoptosis and necrosis in the J774A.1 or THP-1 cells during infection with different leptospires for the indicated times, determined by flow cytometry. The Annexin V + /PĪ cells represent early-apoptotic death while the Annexin V + /PI + cells represent late-apoptotic or necrotic death. The images at “0 h” indicate the early or late apoptosis of the normal and P 2 X 7 -depleted J774A.1 or THP-1 cells before infection. EGTA is an extracellular Ca 2+ chelator, BAPTA/AM is an intracellular free Ca 2+ chelator. Neomycin is a blocker to inhibit IP 3 production. U73122 is a mammalian cell PI-PLC inhibitor. (B). Statistical summary of early or late apoptotic/necrotic ratios in macrophages during infection with different leptospires. Data from experiments such as shown in A. Bars show the means ± SD of three independent experiments. The values at “0 h” indicate the early or late apoptosis of the normal or P 2 X 7 -depleted J774A.1 or THP-1 cells before infection. Five thousand cells were analyzed for each of the samples. *: p

Article Snippet: To determine the source of intracellular free Ca2+ in J774A.1 or THP-1 cells (105 per well) during infection with L. interrogans strain Lai (107 ), the two macrophage monolayers were pre-treated with 2 mM EGTA (Sigma), 100 µM BAPTA/AM (Sigma), 1.2 mM neomycin (Sigma), 20 µM SKF96365 (Sigma), 100 µM verapamil (Sigma) or 10 µM mibefradil (Sigma) for 30 min at 37°C – .

Techniques: Infection, Flow Cytometry, Cytometry, Planar Chromatography

Journal: PLoS ONE

Article Title: Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death

doi: 10.1371/journal.pone.0075652

Figure Lengend Snippet: Elevation of [Ca 2+ ]i in macrophages during infection with L. interrogans . (A). Elevation of [Ca 2+ ]i in J774A.1 and THP-1 cells during infection with L. interrogans strain Lai for the indicated times, determined by laser confocal microscopy. The intensity of green fluorescence reflects the [Ca 2+ ]i in macrophages. The dead strain Lai was obtained by heat killing the spirochete. EGTA is an extracellular Ca 2+ chelator. BAPTA/AM is an intracellular free Ca 2+ chelator. Neomycin is a blocker of IP 3 production. SKF96365 is a receptor-gated calcium channel blocker. Verapamil or mibefradil is L-type or T-type voltage-gated calcium channel blocker. The images at “0 h” indicate the [Ca 2+ ]i in J774A.1 and THP-1 cells before infection. (B). Statistical summary of the [Ca 2+ ]i changes in the leptospire-infected macrophages. Data from experiments such as shown in A. Bars show the means ± SD of three independent experiments. The dead strain Lai corresponds to the heat-killed spirochete. The values at “0 h” indicate the [Ca 2+ ]i in J774A.1 or THP-1 cells before infection. Five hundred cells were analyzed for each of the samples. *: p

Article Snippet: To determine the source of intracellular free Ca2+ in J774A.1 or THP-1 cells (105 per well) during infection with L. interrogans strain Lai (107 ), the two macrophage monolayers were pre-treated with 2 mM EGTA (Sigma), 100 µM BAPTA/AM (Sigma), 1.2 mM neomycin (Sigma), 20 µM SKF96365 (Sigma), 100 µM verapamil (Sigma) or 10 µM mibefradil (Sigma) for 30 min at 37°C – .

Techniques: Infection, Confocal Microscopy, Fluorescence

Journal: Mediators of Inflammation

Article Title: Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK β-AMPK-SIRT1 Signaling Pathway

doi: 10.1155/2016/6152713

Figure Lengend Snippet: Activation of CaMKK β mediates the inhibition of calcium-dependent IL-2 production in RAW 264.7 cells. (a) Cells were treated with LPS in normal DMEM, calcium-free DMEM, calcium-free DMEM with 2 mM CaCl 2 , or DMEM with 5 mM EGTA for 30 min. Protein levels of CaMKK β and pCaMKK β were detected by western blot. Comparison was made for pCaMKK β /CaMKK β . ∗∗ P

Article Snippet: Cell Treatment For calcium modulation, macrophages were treated in calcium-free DMEM (GIBCO) or DMEM with EGTA (Sigma, USA) for calcium deprivation.

Techniques: Activation Assay, Inhibition, Western Blot

Journal: Mediators of Inflammation

Article Title: Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK β-AMPK-SIRT1 Signaling Pathway

doi: 10.1155/2016/6152713

Figure Lengend Snippet: AMPK is activated downstream of CaMKK β for the negative control of the LPS induced IL-12 production in RAW 264.7 cells. (a) Cells were treated with LPS alone or with calcium-free DMEM, 2 mM CaCl 2 , 5 mM EGTA, or 1 μ M STO-609 for 30 min. Protein levels of AMPK α and pAMPK α were detected by western blot. ∗∗ P

Article Snippet: Cell Treatment For calcium modulation, macrophages were treated in calcium-free DMEM (GIBCO) or DMEM with EGTA (Sigma, USA) for calcium deprivation.

Techniques: Negative Control, Western Blot

Journal: Mediators of Inflammation

Article Title: Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK β-AMPK-SIRT1 Signaling Pathway

doi: 10.1155/2016/6152713

Figure Lengend Snippet: LPS induced extracellular calcium influx and modulation of calcium influx affects IL-12 production in murine peritoneal macrophages. (a, b) Macrophages were incubated with LPS (1 μ g/mL) or LPS plus calcium-free DMEM, 5 mM EGTA, 5 μ M SKF96365, or 100 μ M ATP for 6 min. The fluorescence ratio (340 nm/380 nm) of Fura-2AM was captured by using the plate reader assay every 10 s ( n = 4). (c) Macrophages were treated with 5 μ M SKF96365 (SKF) or 100 μ M ATP for 1 h and then stimulated with 100 ng/mL LPS. Supernatants were collected 24 h later and IL-12 p40 and IL-12 p70 levels were detected by ELISA. ∗∗ P

Article Snippet: Cell Treatment For calcium modulation, macrophages were treated in calcium-free DMEM (GIBCO) or DMEM with EGTA (Sigma, USA) for calcium deprivation.

Techniques: Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Bile Salt-Stimulated Lipase from Human Milk Binds DC-SIGN and Inhibits Human Immunodeficiency Virus Type 1 Transfer to CD4+ T Cells

doi: 10.1128/AAC.00593-06

Figure Lengend Snippet: Differences in the DC-SIGN binding capacities of human milk samples from three mothers (S1 to S3). (A) The DC-SIGN-Fc binding capacity was measured for three different human milk samples (1:200). Preincubation of DC-SIGN-Fc with AZN-D1 and EGTA controlled

Article Snippet: DC-SIGN-Fc binding specificity was determined by preincubation of the DC-SIGN-Fc with either 50 μg/ml DC-SIGN-specific mouse antibody AZN-D1 ( ) or 10 mM EGTA (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 20 min before the addition of DC-SIGN-Fc to the coated human milk.

Techniques: Binding Assay