ML 171

Cell experiment:

HT29 cells are cultured in 150 mm diameter plate and when 70-80% confluence is reached, cells are trypsinized, harvested in HBSS and counted. 4×104 cells are dispensed into individual wells in 30 μL final volume (384 well plates) by using a robotic liquid handler. Cells are treated for 60 min at 37°C with 50 nL of DPI, DMSO and library compounds (including ML171) which are automatically dispensed into individual wells from their respective assay plates. This will correspond to a final concentration of 10 μM DPI or library compounds (ML171), and 0.1% DMSO. 20 μL of a mixture containing 200 μM luminol plus 0.32 units of HRP (final concentration) is added. Luminescence is quantified using a 384-well plate luminometer. The data output consisting of the emission intensities for each well is imported into a spread-sheet program (such as Excel) for further processing. As designed, compounds that inhibit Nox1 activity will reduce cellular ROS production, leading to reduced probe-ROS interactions and reduced well luminescence. Compounds are considered ‘hits’ and further processed when light emission is blocked >75% 7 than DMSO wells (DMSO and DPI wells are set to 0% and 100% respectively). Compounds are tested in singlicate at a concentration of 10 μM[1].

References:

[1]. Gianni D, et al. A novel and specific NADPH oxidase-1 (Nox1) small-molecule inhibitor blocks the formation of functional invadopodia in human colon cancer cells. ACS Chem Biol. 2010 Oct 15;5(10):981-93.