FIGURE 5.

Effects of PDMP treatment on the sphingolipid composition and on the in vitro cell motility of A2780/HPR cells. A2780/HPR cells were treated with the specific glucosylceramide synthase inhibitor d-PDMP to achieve sphingolipid depletion. The ineffective stereoisomer l-PDMP has been used as a negative control. Panel A, sphingolipid patterns of A2780/HPR cells untreated (CTR), treated with 10 μm and 20 μm l-PDMP or treated with 10 and 20 μm d-PDMP for 5 days. Cell lipids were extracted with chloroform/methanol/water, 2:1:0.1 by volume, subjected to a two-phase partitioning, and aqueous (left panel) and organic phases (right panel) lipids were analyzed by HPTLC, using as solvent system chloroform/methanol/water, 55:20:3 by volume (spray reagent, aniline/diphenylamine), and chloroform/methanol/0.2% aqueous CaCl₂ 50:42:11 (spray reagent, p-dimethylaminobenzaldehyde), respectively. The equivalent to 500 μg (for organic phases) and 1 mg (for aqueous phases) of cell proteins were loaded on each lane. PE, phosphatidylethanolamine. Panel B, effect of PDMP treatment on in vitro motility of A2780/HPR cells has been assessed by a phagokinetic gold sol assay as described in the legend of Fig. 1. A2780/HPR cells were treated with 10 or 20 μm of l- or d-PDMP for 48 h before the assay, and compounds were maintained in the medium for the whole duration of the assay. The areas of the tracks cleared by the cells have been recorded at time 0 and after 24 and 48 h. Average track areas (means ± S.D. of 50 measurements) normalized for the different cell size are reported in the bar graph. *, p < 0.002 versus time-matched control, °, p < 0.01 versus time-matched l-PDMP-treated cells.