In vitro activity: ML141 enhances the ability of TMX to suppress BLBC cell growth through both induction of cell death and suppression of cell division. ML141 also significantly protects neuroblastoma cells from metformin-induced apoptosis. Moreover, ML141 diminishes K. pneumoniae invasion in a dose-dependent manner.

Kinase Assay: Wild-type GST-Cdc42 (4 μM) is bound to GSH-beads overnight at 4°C. Cdc42 on GSH-beads is depleted of nucleotide by incubating with 10 mM EDTA containing buffer for 20 min at 30°C, washing twice with NP- HPS buffer, then re-suspended in the same buffer containing 1 mM EDTA/or 1 mM MgCl2, 1 mM DTT and 0.1% BSA. Cdc42 unbound sites are blocked by incubation of protein–bead complex for 15 min at RT. Thirty μL of this suspension is incubated with 20 mM inhibitor for 3 min at RT and added 30 μL of various concentrations of ice cold BODIPY-FL-GTP. Samples incubated at 4° C for 45 min and binding of fluorescent nucleotide to enzyme is measured using an Accuri flow cytometer. Raw data are exported and plotted using GraphPad Prism software.

Cell Assay: Cells [Basal-B (MDA-MB 231 and HCC38) and Basal-A with HER2 amplification (HCC1954) cells] are incubated with 500 nM Calcein-AM and 1 μM PI for 15 min, after which live cells and dead cells (represented by positivity of Calcein-AM and PI staining, respectively) are counted utilizing the adherent cell Celigo(TM) cytometer.

Oncotarget. 2014 Nov 30;5(22):11709-22; EMBO Mol Med. 2013 May;5(5):723-36.