Fig. 2.

Reduced GM1 ganglioside and cholesterol in CFTR-silenced cells. A: confluent monolayers of control and CFTR-silenced cells were labeled with cholera toxin B subunit (CtxB) to detect GM1 ganglioside (top) or filipin to detect unesterified cholesterol (bottom). Images were processed identically for the two micrographs shown for each fluorophore so that intensities can be compared. Note decreased overall filipin and CtxB labeling and increased punctate fluorescence in CFTR-silenced cells. Bar, 10 μm. B: GM1 levels in cells were measured biochemically by lipid extraction followed by thin layer chromatography (TLC), detection of gangliosides with resorcinol, and quantitation by image analysis. GM1 was identified by comparison to known standards. Results are expressed relative to control levels. C: total unesterified cholesterol levels in cells were measured biochemically using a cholesterol oxidase-based assay. In B and C, values are means ± SE for 3 samples. *P < 0.05, significantly different in two-tailed t-tests. D: control, CFTR-silenced cells, and CFTR-silenced cells treated for 2 days with 5 μM GM1 ganglioside were extracted, and lipids were separated by TLC. GM1 was detected by CtxB-horseradish peroxidase staining of TLC plates.