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. 2010 Sep;51(9):2527–2545. doi: 10.1194/jlr.M003392

Fig. 10.

Fig. 10.

Giardial GCS is upregulated during parasite stage conversion. A: Microarray analysis at 7 h encystation showing upregulation of the sphingolipid synthesis enzymes GCS and serine palmitoyltransferase 2 compared with trophozoites. Upregulation of cyst wall proteins is shown as positive control. Gene loci are the following: cyst wall protein 1 (CWP1, GL50803_5638), cyst wall protein 2 (CWP2, GL50803_5435), cyst wall protein 3 (CWP3, GL50803_2421), glucosyceramide synthase (GCS, GL50803_11642), serine palmitoyltransferase 2 (SPT2, GL50803_14374), fatty acid elongase 1 (FAE1, GL50803_92729). B: Semiquantitative real-time PCR indicating the upregulation of GCS and CWP1 at 7 h encystation (EN) compared with trophozoite samples (T). Gene expression levels were given as values in arbitrary units relative to the amount of the constitutively expressed house-keeping gene actin. Data are average ± SE (n = 3) of a representative from two experiments done in triplicate. C: Isolated trophozoites (T) or parasites encysted for 24 h (EN) were labeled with 4 μCi/ml of [3H]palmitic acid for 3 h in supplemented PBS. Lipid aliquots corresponding to equal protein amount were separated by 2D-HPTLC using the solvent systems A. Glucosylceramide (GC) levels were expressed as percentage of trophozoite levels; data are average ± SE (n = 3) of a representative from three experiments done in triplicate. D: LC-MS analysis of glycosphingolipids isolated from trophozoites (T) and 24 h encysted cells (EN). GlcCer, glucosylceramide; LacCer, lactosylceramide, GM3 ganglioside (sialic acid, galactosyl-, glucosylceramide), GD3 ganglioside (sialic acid, sialic acid, galactosyl-, glucosylceramide); GGGCer, ceramide trihexoside. Data are average ± SE (n = 3) of a representative from two experiments done in triplicate. E: Parasites expressing recombinant giardial GCS or an empty plasmid (cntl) were encysted for 16 h and stained for CWP1. Cyst number is presented as percentage of total cell number (TOT) ± SE (n = 3) of a representative from three experiments done in triplicate.