Glucosylceramide synthase assay

GCS activity was assayed as described previously [60, 61], with modification. Briefly, cells were grown 24 hr in 35-mm dishes (5×10⁶ cells/dish) in 10% FBS RPMI-1640 medium, and then switched to 1% BSA RPMI-1640 medium containing 2.0 μM 7-nitro-2,1,3-benzoxadiazole (NBD) C6-ceramide complexed to BSA (Invitrogen). After 2 h incubation at 37°C, cellular lipids were extracted and reconstituted with chloroform/methanol (1:1, v/v; 200 fluorescence units/100 μl). From this preparation, a 5-μl of portion was loaded by autosampler onto a normal-phase silica column (ZORBAX Rx-SIL, 5 μm, 4.6 × 250 mm). NBD sphingolipids were eluted by linear gradient (0-14 min, 1 ml/min) using solvent system A (chloroform/methanol/ortho-phosphoric acid) (80:20:0.1, v/v/v) and solvent system B (chloroform/methanol/H₂O/ortho-phosphoric acid) (60:34:6:0.1, v/v/v/v), and detected with an Agilent 1260 fluorescence detector (λ_excitation= 470 nm, λ_emission = 530 nm) (Agilent, Santa Clara, CA; HPLC system: Agilent 1220 Infinity LC Gradient System VL). Individual sphingolipids were identified and quantitated with a separate standard curve for each. Each sample was analyzed at least three times, and GCS activity is defined in terms of glucosylceramide produced in 2 hr normalized against total cellular proteins. To assess tumor GCS activity, tumors were pieced (<1 mm) and digested with collagenase IV for 2 hr, and samples were then prepared and processed as described above.