Fig 1.
Ceramides with different amide-linked acyl chains and cholesteryl phosphocholine used in this study.
Fig 2.
(A) The amounts of radiolabeled C6-, C10- and C16-Cer uptake as nmol ceramide per mg total protein as a function of time. HeLa cells were labeled with [3H]Cer (50 μM), with the radionuclide in positions 4 and 5 of the sphingosine backbone. The uptake of ceramide by HeLa cells was analysed by HPTLC and scintillation counting. (B) Amount of ceramide precursor incorporation into HeLa cells at different loading concentrations of ceramides after a 3-hour incubation. The data for the incorporation of the radiolabeled [3H]Cers are from at least three different experiments.
Fig 3.
Radiolabeled ceramides with different acyl chain lengths metabolize into sphingolipids as a function of time.
HeLa cells were loaded with C6-Cer (A), C10-Cer (B) and C16-Cer (C) in CholPC complexes. The [3H]sphingosine labeled ceramides (50 μM) were allowed to be taken up by HeLa cells for 3, 6 and 24 hours, and the incorporation was followed by HPTLC analysis. The amount of each lipid is presented as a percentage of the total radioactivity in the sample. GSLs = GlcCer, GalCer, GlcCer-OH and GalCer-OH. Other = the remaining lane. The data is from at least three different experiments.
Fig 4.
HeLa cells C6- and C10-Cer precursor uptake.
Analysis of (A) C6-Cer and (B) C10-Cer incorporation at different concentrations. The ceramides were [3H]-labeled in the sphingosine part. After 24 h of ceramide uptake the total lipids were extracted and analysed by HPTLC. “Start” and “Front” refer to the labeled lipids remaining on the application spot, or that were eluted along with the solvent front, “Other” refers to the traces of radiolabeled lipids between the identified spots (see S1 Fig). The data is from at least three different experiments.
Fig 5.
Analysis of C16-Cer precursor incorporation at different concentrations.
The ceramide was either (A) [3H]-labeled in the sphingosine backbone or in the (B) palmitic acid portion. After 24 h of ceramide uptake the total lipids were extracted and analysed by HPTLC. “Start” and “Front” refer to the labeled lipids remaining on the application spot, or that were eluted along the solvent front, “Other” refers to the traces of radiolabeled lipids between the identified spots (see S1 Fig). The data is from at least three different experiments.
Fig 6.
Cytotoxic effect of ceramides.
HeLa cells were treated with increasing concentrations of C6-, C10-, C16-Cer or cholesterol, complexed with CholPC, for 22 h and compared to vehicle (PBS) treated cells. Subsequently, a resazurin reduction assay was performed. Viability of the HeLa cells is represented by mean ± SEM of at least 3 independent experiments and is expressed as percentage survival of PBS control.