Erythro 9 2 hydroxy 3 nonyl adenine

To estimate the contribution of microtubules, actin and cytoskeletal motor proteins their specific inhibitors were applied. Microtubule depolymerization was induced by 10 nM nocodazole (Sigma-Aldrich), whereas microtubules were stabilized by 0.5 nM taxol (Sigma-Aldrich). Actin dynamics was influenced by 100 nM of cytochalasin D (Sigma-Aldrich). Myosin II activity was blocked by 10 μM blebbistatin (-/-, Sigma-Aldrich), whereas dynein was inhibited using 0.5 mM EHNA (erythro-9-(2-hydroxy-3-nonyl)-adenine, Sigma-Aldrich). In these inhibition experiments, C6 and U87 cells were seeded in patterned (stamped) 6-well tissue culture plates (Greiner), and allowed to spread for 3 hours. Inhibitors were added shortly before imaging, and were kept on the cells for the duration of the experiments. Cells were imaged for at least 14 hours in the presence of either a solvent control (DMSO (Sigma-Aldrich) or water) or one of the indicated drugs.

Glyceryl triacetate, buffer reagents, β-glycerophosphate, erythro-9-(2-hydroxy-3-nonyl) adenine, α, β-methyleneadenosine 5’-diphosphate, and 2-mercaptoethanol were purchased from Sigma (Sigma, St. Louis, MO, USA). A mouse anti-human CD73 antibody and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody were obtained from AbD Serotec (AbD Serotec, Raleigh, NC, USA). A HRP-conjugated goat anti-mouse IgG antibody was purchased from Jackson ImmunoResearch (Jackson ImmunoResearch, Westgrove, PA, USA). A mouse anti-adenosine receptor A_2A antibody was obtained from Upstate (Upstate, Billerica, MA, USA). A goat anti-adenosine kinase antibody, mouse anti-α tubulin antibody, HRP-conjugated donkey anti-goat IgG antibody, and HRP-conjugated goat anti-mouse IgM antibody were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Absolute ethanol was from Pharmco (Pharmco, Brookfield, CT, USA).