Fluorescent Assay for Amidase Activity

Cells were plated in 96-well plates at 20,000 cells per well with 25 μl in the appropriate medium (see above). SACLAC, SOCLAC, and DMSO were prepared in medium supplemented with 20% FBS at the appropriate concentrations from a DMSO stock solution, and 25 μl were dispensed onto the 96-well plate. Cells were incubated with the indicated concentrations of SACLAC, URB597, or DMSO (vehicle control) for 1 h or the indicated times in a humidified incubator at 37°C and 5% CO₂. For time-course studies, cells were resuspended in inhibitor-free culture medium before RBM1-151 incubation. Unless indicated otherwise, after incubation with the inhibitors, 50 μl of RBM1-151 was dispensed onto the cells at a final concentration of 20 μM in medium with 20% FBS. Cells were incubated with RBM1-151 for 1 h in a humidified incubator at 37°C and 5% CO₂. After RBM1-151 incubation, 25 μl of 100% methanol was added to each well. Immediately after, 100 μl of 2.5 mg/ml sodium periodate in 100 mM glycine (pH 10.6) was added to each well. The plate was incubated in a humidified incubator at 37°C and 5% CO₂ for 30 min. Fluorescence was measured at 355 nm excitation and 460 nm emission using a microtiter plate reader. During data analysis, background signal from RBM1-151 in culture media without cells was subtracted from all values, and each amidase activity was calculated using the following equation: [umbelliferone]_C - [umbelliferone]_I, where [umbelliferone] corresponds to the amounts in μM/h/2 × 10⁴ cells produced in both control cells (C, treated with DMSO) and cells treated with each inhibitor (I) calculated from an umbelliferone calibration curve.