Figure 1.
The oxidized phospholipids POVPC and PGPC are cytotoxic and induce apoptosis.
(A) Chemical structures of POVPC and PGPC. Dotted circle represents the functional group at sn-2 position. (B) RAW 264.7 cells were incubated with 50 µM POVPC and PGPC for indicated periods. Control cells were treated with 1% ethanol. Cell viability was determined by Vybrant® MTT assay kit. Results are expressed as a percentage of viable cell number in treated cells compared with that of untreated control cells. Data are means ± S.D., n = 8 in each group. (C) Cells were incubated with the stated concentrations of POVPC and PGPC for 4 h. The cells were analyzed for Alexa Fluor488-annexin V and propidium iodide fluorescence staining by Flow cytometry as described under “Experimental Procedures”. Results are represented as means ± S.D. Probabilities compared to control were determined by Student’s t-test (two-tailed, unpaired); ***p < 0.001,(n = 8 in each group).
Figure 2.
CerS expression and endogenous CerS activity in RAW 264.7 cells.
(A) RAW 264.7 cells were harvested and cDNA was synthesized. CerS mRNA levels were measured by RT-qPCR as described under “Experimental Procedures”. The data are normalized to GAPDH mRNA expression and data are means ± S.E. for three independent experiments performed in triplicate. (B) CerS activity was assayed in cell homogenates using C16-CoA, C18-CoA, C20-CoA and C22-CoA substrates as described under “Experimental Procedure”. Results are means ± S.E. for four independent experiments.
Figure 3.
Influence of POVPC and PGPC on ceramide levels and CerS activation in RAW 264.7 cells.
(A) Both POVPC and PGPC elevate ceramide generation in RAW 264.7 cells. Cells were stimulated for 24 h with respective OxPLs (50 µM) in parallel to ethanol treated control cells. Lipids were extracted and analyzed for ceramide levels by LC/MS-MS as described under “Experimental Procedure”. No probability. values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (B) Ceramide speciation was performed after OxPL treatment as above. The data are means ± S.E., *p<0.05, **p < 0.01 compared with control, n = 4. (C) After OxPL treatment cells were harvested and CerS activity in cell homogenates was measured as described earlier. Results are means ± S.E., *p < 0.01, **p < 0.05, of a typical experiment repeated four times with similar results. (D) CerS mRNA levels were measured by RT-qPCR after 24 h incubation with 50 µM POVPC or PGPC as described under “Experimental Procedures”. The data are normalized to GAPDH mRNA expression and data are means ± S.E. for four independent experiments performed in triplicate. (E) nSMase activity was measured in cell homogenates after exposure to OxPL as described under “Experimental Procedures”. The data are represented as means ± S.E., n = 4.
Figure 4.
Effect of FB1 on OxPL induced CerS activation and ceramide levels.
(A) Raw 264.7 cells were pre-incubated with FB1 (20 µM) for 2 h prior to the 24 h OxPL treatment. Lipids were extracted and ceramide levels were analyzed as described under “Experimental Procedure”. No probability values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (B) Ceramide species were analyzed as earlier. The data are means ± S.E., *p < 0.05, **p < 0.01 compared with control, n = 4.
Figure 5.
Effect of lipid extracts from LDL and OxLDL on CerS activity.
(A) Oxidation of LDL was performed as described under “Experimental Procedure”. RAW 264.7 cells were stimulated with lipid extracts from native LDL and OxLDL (50 µg protein/mL respectively) for 24 h. CerS activity in cell homogenates was measured using C16-CoA and C22-CoA substrates. Results are means ± S.D., *p < 0.05, of a typical experiment repeated four times with similar results. (B) Cells were treated with intact LDL and OxLDL (50 µg protein/mL respectively) as described above for 24 h. CerS activity in cell homogenates was measured using C16-CoA and C22-CoA substrates. Results are means ± S.D. *p < 0.05, of a typical experiment repeated four times with similar results. (C) Cells were treated as described above and lipids were extracted and analyzed for ceramide content as described under “Experimental Procedure”. No probability values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (D) Ceramide species were analyzed after treatment with lipid extracts from native LDL and OxLDL as described earlier. The data are means ± S.E., *p < 0.05, n = 4.