Figure 1.
TLC analysis of metabolically labeled GSLs.
(A) Metabolic labeling of HeLa cells, normal (black), GLTP siRNA (red) and overexpression of GLTP (green). 3H-sphinganine incorporation into the sphingolipids was analyzed using TLC, and a mixture of both gene sequence #76 and #78 of the siRNA GLTP constructs were used. The TLC data for the incorporation of the radiolabeled 3H-sphinganine are from at least three different experiments, and the results are normalized to the controls. (B) Representative TLC plates illustrating a typical separation of the analyzed lipids. The plates were first stained with the carbohydrate specific orcinol, followed by 3% Cu acetate charring to visualize all lipids. Left plate, shows the effective separation of GlcCer, GalCer and LacCer using the solvent system 10∶2:4∶2:1 chloroform:methanol:acetone:acetic acid:H2O (v/v). Right plate, for effective Gb3 separation from other GSLs we used the 45∶55:10 chloroform:methanol:0.2% CaCl2 (in H2O) (v/v) solvent system. The statistical significance compared to the respective controls is indicated with asterisks. One asterisk (*) p<0.05 and two asterisks (**) p<0.01 indicate the statistical significance compared to the controls.
Figure 2.
Analysis of metabolically labeled PC, SM and PE after alternation in the GLTP expression levels.
Three phospholipid fractions were analyzed using 3H-acetate incorporation of HeLa cells with different expression levels of GLTP. The TLC data for the incorporation of the radiolabeled 3H-acetate are from at least three different experiments, and the results were normalized to the controls. The statistical significance compared to the respective controls is indicated with asterisks. Two asterisks (**) p<0.01 indicate the statistical significance compared to the controls.
Figure 3.
Relative change in GlcCer/Galcer, LacCer and Cer after knockdown or overexpression of GLTP in HeLa cells normalized to control cells.
(A) Changes in the total masses of GlcCer/GalCer and GlcCer/GalCer with d18∶0 and d18∶1 base respectively. (B) Changes in the GlcCer/GalCer species after siRNA or OE treatments, compared to the control. (C) Changes in the total masses of LacCer and LacCer with d18∶0 and d18∶1 base respectively and (D) changes in the LacCer species. (E) Changes in the total masses of Cer and Cer with d18∶0 and D18∶1 base respectively, with (F) showing the changes in the Cer species. The data for siRNA GLTP (red bars) and GLTP OE (green bars) were normalized to the levels in the mock control cells (black bars). The abbreviations for the lipid classes are given in “Materials and Methods”. For clarity, minor species are not presented in the graph. For full list of species and quantitative data with SD, see Table S1).
Figure 4.
Relative change in Gb3, CE and DAG after knockdown or overexpression of GLTP in HeLa cells compared to control cells.
(A) Changes in total (left side) and molecular species of Gb3 in HeLa cells subjected to knockdown (red bars) or overexpression (green bars) of the GLTP gene normalized to the levels in the mock control cells (black bars). The Gb3 level for the GLTP siRNA and OE sample is significantly different (p<0.01) than the control sample. (B) Relative changes in the total and molecular species of CE’s and (D) DAG’s as a function of GLTP expression levels. The abbreviations for the lipid classes are given in “Materials and Methods”. For clarity, minor species are not presented in the graph. For full list of species and quantitative data with SD, see Table S2.
Figure 5.
Relative changes in SM, PC and ether-PC after knockdown or overexpression of GLTP in HeLa cells compared to control cells.
The relative changes in the masses of (A) SM and (B) PC in HeLa cells subjected to knockdown (red bars) or overexpression (green bars) of the GLTP gene normalized to the levels in the mock control cells (black bars). The changes for the ether linked PCs is on the far right, yellow represent the GLTP siRNA sample and the blue bar the GLTP OE sample. The abbreviations for the lipid classes are given in “Materials and Methods”. For clarity, minor species are not presented in the graph. For full list of species and quantitative data with SD, see Table S3.
Figure 6.
Relative changes in phosphoglycerides after knockdown or overexpression of GLTP in HeLa cells compared to control cells.
Changes in the total masses and the molecular species of (A) PE, (B) PS, (C) PI and (D) PG in HeLa cells subjected to knockdown (red bars) or overexpression (green bars) of the GLTP gene normalized to the levels in the mock control cells (black bars). The abbreviations for the lipid classes are given in “Materials and Methods”. For clarity, minor species are not presented in the graph. For full list of species and quantitative data with SD, see Table S4.
Figure 7.
(A) Comparison of the total lipid content summarized (from the MS data) between CTRL (black bars), GLTP siRNA cells (red bars) and GLTP overexpressing cells (green bars). Lipid quantities were calculated on the basis of the corresponding IS and the amounts (pmol/500000 cells) represented the sum of all individual molecular species of all lipid classes. (B) Comparison of the abundance of the lipid classes analyzed in this study. (C) Changes in the distribution of the amide-linked hydrocarbon chains in sphingolipids (GlcCer/GalCer, LacCer, Gb3, Cer and SM) of normal, GLTP siRNA and GLTP overexpressing HeLa cells. (D) Changes in the degree of unsaturation of the acyl chains in the different phospholipid classes relative to GLTP expression. Left control, middle GLTP siRNA and right GLTP OE HeLa cell samples, n is the sum of degree of unsaturation of fatty acid chains in the phospholipids.