Figure 5

Apoptotic effects of oxPL in RAW264.7 cells and BMM. Cells were incubated with the indicated concentrations of oxPLs in media under low serum conditions (0,1% FCS) for 4 h and analyzed by flow cytometry (see materials and methods). The fraction of apoptotic cells was determined from Annexin V staining of externalized phosphatidylserine. Control cells were incubated with 1% v/v Ethanol (EtOH). 2,5 mM H₂O₂ or 1 μM staurosporin (STS) in the incubation media, which were used as reference agents inducing necrosis or apoptosis, respectively. Panel A: 50 μM POVPC and PGPC induce apoptosis in RAW264.7 macrophages. PGPC is a more potent inducer of cell death than POVPC under these conditions. Results are means of 6 replicates out of three independent experiments. Panel B: 50 μM PGPC and POVPC induce apoptosis in BMM, which are slightly more sensitive to the oxPL compared with RAW264.7 macrophages. Results are means from one representative experiment of 4 replicates of one pooled sample (BMM isolated and pooled from femurs and tibias of 5 mice, see materials and methods). Panel C: 50 μM 1-O-alkyl ether analogs of PGPC and POVPC (E-PGPC and E-POVPC, respectively) induce apoptosis in RAW264.7 macrophages. Panel D: Representative histogram of RAW264.7 cells after exposure to 1% v/v Ethanol (EtOH), 1 μM staurosporin (STS), POVPC or PGPC. Experimental conditions as described under Panel A. All Results are expressed as means +/− SD. Significance was determined by Student’s t-test (two tailed, unpaired). ** p ≤ 0,01, *** p ≤ 0,005.