K 3200

The enzymatic activity of acid sphingomyelinase (SMPD1/ASM) was measured by the cleavage of HMU-PC using a commercial kit (Echelon, K-3200) as described previously (van Diggelen et al., 2005 (link)). GBM cells with indicated hours DMSO or fluoxetine treatment were collected. Cell pellets were then resuspended in water with proteinase inhibitor and sonicated in an ice water bath for 10 cycles (30 s on and 30 s off). Tumor samples were first homogenized in water with proteinase inhibitor by a rotor-stator tissue homogenizer and then sonicated on ice. After 5-minutes centrifugation (10,000 × g) at 4°C, protein concentration of each sample was determined. Equal volumes of 10 μg samples were added into each reaction and incubated at 37°C for 3 hours. After adding the stop buffer, the fluorescence of HMU was recorded on an Infinite M1000 Plate Reader (Tecan) at 360 nm excitation and 460 nm emission. Data were normalized to that of the indicated control group and plotted from four biological replicates.

ASMase activity was measured fluorometrically (k-3200; Echelon Biosciences Inc.) according to the manufacturer’s instructions with the following modifications: Assays were performed on live pHBE cells expressing EGFP-CFTR that had been seeded in fluorodishes as described above. Substrate buffer and ASMase substrate were added onto the cells, and ASMase activity was measured by collecting samples under control conditions and during acute stimulation with VIP (200 nM), with or without Ami pretreatment (13 µM for 1 h). Stop buffer was added to samples taken 30, 60, or 120 min after adding substrate. Fluorescence intensity was measured using a spectrophotometer at 360 nm excitation and 460 nm emission at several time points after adding stop buffer (10, 20, and 30 min) to ensure that fluorescence was stable.