Evidence for the Involvement of Lipid Rafts and Plasma Membrane Sphingolipid Hydrolases in Pseudomonas aeruginosa Infection of Cystic Fibrosis Bronchial Epithelial Cells

Figure 4

CuFi-1 cells overexpressing GBA2 tagged with GFP. (a) Confocal images of CuFi-1 cells overexpressing GFP (CuFi-1 mock) and overexpressing GBA2-tagged GFP (CuFi-1-GBA2). (b) Total and plasma membrane enzymatic activity of GBA2 measured in parental CuFi-1 cells and CuFi-1-overexpressing GBA2 and mock cells. The measurement of the total GBA2 enzymatic activity was conducted on cell lysate and the activity expressed as pmoles of product on mg of proteins per hour. The measurement of the plasma membrane enzymatic activity of GBA2 was conducted on living cells and expressed as pmoles of product on 10⁶ cells per hour. versus mock. On the right mRNA expression of GBA2 in CuFi-1 and GBA2-overexpressing cells evaluated by qRT-PCR, data are expressed as fold increase with respect to control cells. versus CuFi-1. (c) Radioactive sphingolipid composition of CuFi-1-overexpressing GBA2 compared to mock cells. Cell sphingolipids were metabolic labeled at the steady state using [1-³H]-sphingosine as indicated in Materials and Methods. Left: representative digital autoradiography of radioactive lipids of two different clones of CuFi-1-overexpressing GBA2 and in one clone of mock cells. Right: semiquantitative graph of sphingolipid species. Data are reported as percentage of radioactivity associated with sphingolipids. Cer: ceramide; GlcCer: glucosylceramide; PE: phosphatidylethanolamine; Gb3: globotriaosylceramide; SM: sphingomyelin. versus CuFi-1 mock cells

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