BMP1 enzyme activity was measured using a fluorescent assay according to the manufacturer's instructions (R&D Systems). Briefly, cultured NTM cell strains (n = 3) were grown in a 6-well plate and maintained until 100% confluent. Subsequently, TM cells were serum deprived for 24 hours, and then pretreated with the potent and selective BMP1 inhibitor 3-(aminocarbonyl)-β-(3-cyclohexlpropyl)-N-hydroxy-1,2,4-oxadiazole-5-propanamide (UK383367; R&D Systems) at various concentrations (1, 3, or 5 μm) for 1 hour. The medium was changed, and BMP1 inhibitor and TGF-β2 (5 ng/mL) were added for an additional 24 hours. CM was collected and 25 μL of CM were diluted to 50 μL with buffer (25 mM HEPES, 0.1% Brij-35, pH 7.5). Then, 50 μL of the fluorogenic substrate MCA-Tyr-Val-Asp-Ala-Pro-Lys (DNP)-OH (20 μM; R&D Systems) were added and the solution subsequently was loaded into a black well plate. The enzymatic activity was measured using a fluorescent plate reader (M200; Tecan, Durham, CA) at an excitation wavelength of 320 nm and emission wavelength of 405 nm. A standard curve was derived using a standard compound (MCA-Pro-Leu-OH; Bachem, Torrance, CA) for BMP1 enzyme assays.