| In Vitro | In vitro activity: DMOG shows only weakly active in the microsomal system, but efficiently suppresses hydroxyproline synthesis in intact cells. DMOG reduces FGF-2-induced proliferation and cyclin A expression by inhibiting prolyl hydroxylase activity in HPASMC.
Kinase Assay:
Cell Assay: To analyze DNA synthesis as an index of cellular proliferation, VSMC are plated in 48-well plates (5,000 per square centimeter) in growth medium, incubated overnight, and serum-deprived (1% FCS) for 24 h. Replicate wells are then stored at –70°C for baseline (day 0) cell counts, and fresh medium with or without growth factors is added to the remaining wells, which are incubated 72-96 h in 20 or 5% O2. Days 0 and 3 or 4 cell counts are determined by lysing cells in a buffer containing a fluorescent dye, which has minimal fluorescence by itself but fluoresces when bound to DNA or RNA. Absolute cell numbers are calculated by comparing the fluorescence of specimens with that of a standard curve similarly prepared using a known number of cells. |
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