Journal:

Article Title: Lysophosphatidic acid (LPA) and Angiogenesis

doi: 10.1007/s10456-008-9113-5

Figure Lengend Snippet: To analyze the role of LPA receptors in LPA-induced angiogenesis we used the LPA1 and LPA3 receptor antagonist, VPC32183. Using the same approach aforementioned, we show in 3A and 3B that LPA-induced angiogenesis is blocked completely by this compound at 10µM. Importantly, VPC32183 alone (at 10µM) had no discernable effect in angiogenesis and does not block VEGF-induced angiogenesis (Figure 3C). One-Way ANOVA test was performed between groups. ** P< 0.001. Error bars represent standard deviation (SD). In figure 3B (scale bar: 0.125 cm), we present the image of a single membrane representative of each group.

Article Snippet: Disclosures : VPC32183 is sold by Avanti Polar Lipids under license from the University of Virginia.

Techniques: Blocking Assay, Standard Deviation, Membrane

Journal:

Article Title: Lysophosphatidic acid (LPA) and Angiogenesis

doi: 10.1007/s10456-008-9113-5

Figure Lengend Snippet: S-1-oleoyl-2-O-methylglycerophosphothionate (S-OMPT), at 100nM, is able to induce a strong angiogenic response, similar to the LPA response at this concentration (4A–4C). CAMs treated with S-OMPT (100nM) together with VPC32183 (10µM) document that the response is completely blocked by inclusion of this antagonist (4C). For statistical analysis, Student t test was performed between groups. ** P< 0.001. Error bars represent standard deviation (SD). Figure 4B is the image of a single membrane that is representative of each group. Scale bar: 0.125 cm.

Article Snippet: Disclosures : VPC32183 is sold by Avanti Polar Lipids under license from the University of Virginia.

Techniques: Concentration Assay, Standard Deviation, Membrane

Journal:

Article Title: Lysophosphatidic acid (LPA) and Angiogenesis

doi: 10.1007/s10456-008-9113-5

Figure Lengend Snippet: CAMs were treated with recombinant sphingomyelinase D (SMaseD) and the number of vessels that intersected the disc was counted. This enzyme is able to induce an angiogenic response (5A) similar to the LPA response. Two catalytically inactive forms of this enzyme (H37N and H73N) were unable to induce angiogenesis (5B), suggesting that the response obtained is dependent on the catalytic activity (lyso-PLD) of the arachnid enzyme. Moreover, SMaseD-induced angiogenesis is blocked by the LPA receptor antagonist VPC32183 at 10µM (5C), suggesting that the positive response obtained is dependent on the activation of LPA1,3 receptors. One-Way ANOVA test was performed between groups. ** P< 0.001. Error bars represent standard deviation (SD).

Article Snippet: Disclosures : VPC32183 is sold by Avanti Polar Lipids under license from the University of Virginia.

Techniques: Recombinant, Activity Assay, Activation Assay, Standard Deviation

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Vehicle-dependent Effects of Sphingosine 1-phosphate on Plasminogen Activator Inhibitor-1 Expression

doi: 10.5551/jat.37663

Figure Lengend Snippet: S1P bound to albumin induced PAI-1 expression by activating S1PR2 pathway in 3T3L1 adipocytes. (A) 3T3L1 adipocytes were pre-incubated for 18 hours in FBS-free DMEM and the medium was exchanged for FBS-free DMEM containing 20 µM of VPC23019, JTE013, or DMSO for 30 minutes. Then, the cells were challenged with a medium containing 10 µM of sphingosine 1-phosphate (S1P) with the corresponding inhibitors for 4 hours. The mRNA level of plasminogen activator inhibitor 1 (PAI-1) was analyzed using real-time PCR. 18S was utilized as an internal control ( n = 4–6/group). † p < 0.01 vs. DMSO alone, YC1 alone, S1P + VPC, and JTE alone, and p < 0.05 vs. S1P + JTE. ‡ p < 0.01 vs. other groups. (B, C). 3T3L1 adipocytes were pre-incubated for 18 hours in FBS-free DMEM, and the medium was exchanged for FBS-free DMEM with 10 µM of S1P bound to apoM-rich vehicle (ApoM) or control vehicle (Null) for 4 hours. Then, the modulation of the phosphorylation of moesin in 3T3L1 cells was examined using western blotting ( n = 6/group). ** p < 0.01, *** p < 0.001.

Article Snippet: VPC23019 (857360P; Avanti Polar Lipids, Alabaster, AL), JTE013 (10009458; Cayman Chemical, Ann Arbor, MI), Y27632 (257-00511; WAKO Pure Chemical Industries, Osaka, Japan), wortmannin, YC-1 (W1628, Y102; Sigma-Aldrich Co), and SIS3 and BAY11-7082 (sc-222318, sc-200615; Santa Cruz Biotechnology, Inc. TX) were dissolved in DMSO.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Control, Phospho-proteomics, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

doi: 10.1186/s13045-022-01390-5

Figure Lengend Snippet: The αCD33-mAB-P/P nanocarriers only transport siRNA in presence of free SMCC-protamine (SMCC-P). A Coomassie-stained SDS–PAGE showing αCD33-mAB, αCD33-mAB coupled with SMCC-P and HPLC-fractions 29–30 of αCD33-mAB-P/P upon effective depletion of unbound SMCC-P; HC = heavy chain, LC = light chain, -P = SMCC-protamine. B Antibody–protamine conjugates with fluorescent Alexa488-siRNA in cell-free incubation overnight on chamber slides. αCD33-mAB-P/P with free SMCC-P forms visible vesicular structures (left panel), while αCD33-mAB-P after depletion of free SMCC-P (fraction 30, see A) do not form visible structures (right panel). C. DLS and zeta-potential measurement of αCD33-mAB-P/free protamine-scr-siRNA carriers. D. αCD33-mAB-P/P-scr-siRNA nanoparticles were left to form for 2 h and subjected to electron microscopy on copper grids by phosphotungstate negative staining. E–H. Immunostaining of nanocarriers with an anti-human IgG antibody to illustrate the accessibility and location of the αCD33-mAB in the outside rim and the siRNA in the lumen of the αCD33-mAB-P/P-nanocarriers. I. Protamine was chemically coupled to Cy3 and then incubated with αCD33-mAB-P that was depleted from free protamine and with non-fluorescent control-siRNA to form nanocarriers. This complexation was performed for 2 h at RT and nanocarriers were then immobilized o/n on slides for immunostaining as in G. J. αCD33-mAB-P/P-Cy3-control-siRNA show homogeneous Cy3 (blue) micelles. K. The same vesicles as in L show anti-human IgG-Alexa647 (red) fluorescence in ring-like structure around each vesicle (staining as depicted in E). L. Overlay of panels J and K. α, anti

Article Snippet: Protamine was conjugated using Cy3 Mono-Reactive Dye Pack (cat. no. PA23001, Cytiva, Freiburg, Germany) according to the manufacturer’s protocol to achieve protamine-Cy3.

Techniques: Staining, SDS Page, Incubation, Zeta Potential Analyzer, Electron Microscopy, Negative Staining, Immunostaining, Control, Fluorescence

Journal: Journal of Hematology & Oncology

Article Title: Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

doi: 10.1186/s13045-022-01390-5

Figure Lengend Snippet: Cellular targeting of Bruton’s kinase (BTK) by αCD33-mAB-P/P-ibrutinib-Cy3.5 and inhibition of clonal growth of treated AML-cells. A Schematic overview: spontaneous assembly of the αCD33-mAB-P/P-ibrutinib-Cy3.5 nanocarrier. B The αCD33-mAB-P/P conjugate was incubated for 2 h with anionic ibrutinib-Cy3.5 (left panels) or uncharged ibrutinib (trademark: imbruvica™; right panels) in 1:20 ratio and applied to cell-culture treated glass slides for fluorescence microscopy. Only αCD33-mAB-P/P-ibrutinib-Cy3.5 complexes led to the formation of numerous vesicles, where the larger vesicles showed intense Cy3.5 fluorescence (upper left panel) and vesicle formation in phase contrast (PC, lower left panel). No nanocarrier formation in presence of uncharged ibrutinib (upper and lower right panels, bubbles in lower right panel are mounting air inclusion artifacts). C Electromobility shift assays showing the electrostatic loading capacity of ibrutinib-Cy3.5 to conjugates from A in a molar ratio. One mol of αCD33-mAB-P/P can bind at least 20–50 mol ibrutinib-Cy3.5. D CD33-positive OCI-AML2 cells were treated by the respective conjugates shown for 72 h, lysed and subjected to SDS–PAGE and Western blotting for phospho-BTK (pBTK), total BTK (tBTK) and actin as a loading control. Both, free ibrutinib-Cy3.5 and αCD33-mAB-P/P-ibrutinib-Cy3.5 complexes inhibited the phosphorylation of BTK. E-M Fluorescence microscopy of OCI-AML2 cells treated with targeting conjugates and controls showing a marked intracellular enrichment of Cy3.5-signals (I). Fluorescence microscopy of OCI-AML2 cells pre-treated with ibrutinib-bodipy (green, G and M) do not show intracellular enrichment of Cy3.5-signals after αCD33-mAB-P/P-ibrutinib-Cy3.5 treatment (J compared to G). N Upper panels: Photographs of representative colony formation assays as summarized in the lower panel. In colony formation assays, 1200 nM untargeted ibrutinib-Cy3.5 did not reduce colony growth of OCI-AML2 cells, while the specifically targeted αCD33-mAB-P/P-ibrutinib-Cy3.5 (60 nM nanocarrier: 1200 nM ibrutinib-Cy3.5) reduced the colony growth to below 30% of the PBS controls, more than the treatment with 1200 nM uncharged ibrutinib (right-most bar). Significance: *, p < 0.05, 2-tailed T-test. Means plus SD of 3 independent experiments. α, anti

Article Snippet: Protamine was conjugated using Cy3 Mono-Reactive Dye Pack (cat. no. PA23001, Cytiva, Freiburg, Germany) according to the manufacturer’s protocol to achieve protamine-Cy3.

Techniques: Inhibition, Incubation, Cell Culture, Fluorescence, Microscopy, SDS Page, Western Blot, Control, Phospho-proteomics