Zardaverine (at single doses of 1.5 mg, 3.0 mg, or 6.0 mg) was administered by metered dose inhaler

Zardaverine and anagrelide were tested in 16-point dose–response, comprising twofold dilutions from a top concentration of 7.5 μM, in a panel of malignant and non-malignant cell lines (CS242 embryonal rhabdomyosarcoma (ERMS), CS242 fibroblast, RD ERMS (ERMS of muscle origin), Rh18 ERMS, Rh36 ERMS, Normal female fibroblast, ERMS derived from paratesticular relapse, T24, HeLa). Compounds were tested in CS242 ERMS and RD ERMS seeded at 1,250 cells/well, and in CS242 fibroblast, normal female fibroblast, Rh18 ERMS, Rh36 ERMS, T24, and HeLa seeded at 313 cells/well. Cells were exposed to compound (0.1-10000nM) for 72 h at 37°C, 5% CO2. To determine cell viability, plates were allowed to cool to room temperature for 1 h after removal from 37°C incubation, 20 μl/well of CellTiter-Glo was added using a MicroFlo Select microplate reagent dispenser (BioTek, Winooski, VT, USA), and luminescence was measured after an additional 30 min at room temperature using an EnVision microplate reader (PerkinElmer, Waltham, MA, USA). Signal stability was confirmed by re-reading luminescence every 10 min for 2 h.