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. 2010 Mar 23;5(3):e9826. doi: 10.1371/journal.pone.0009826

Figure 4. Effects of TNFα and oxLDLs on fro/fro fibroblasts transfected with active V5-nSMase2 vector.

Figure 4

The fro/fro fibroblasts were stably transfected using pEF-6 plasmid containing V5-nSMase2 cDNA. After clone selection, the expression of V5-nSMase2 was evaluated by western blot using an anti-V5 antibody (A), immunocytochemistry (B) and by enzymatic determination of nSMase activity, under basal conditions (C) and after activation by TNFα (50 ng/ml) or oxLDLs (200 µg/ml) (D). Time course of sphingomyelin hydrolysis induced by TNFα (50 ng/ml) or oxLDLs (200 µg/ml) were determined in V5-transfected cells under the conditions indicated in the legend to Figure 2 (E). Caspase activity was evaluated by western blot of caspase-3 (32 KDa) (F), and by fluorometric determination of DEVDase activity (G), under the conditions of Fig. 3. Apoptosis triggered by TNFα (50 ng/ml, 48 h incubation) or oxLDLs (200 µg/ml, 24 h incubation) was evaluated by fluorescence microscopy counting of cells labeled by syto13/PI, under conditions of Fig. 1 and 3 (H). In Fig. 4C–E, and G,H, the data are expressed as mean ± SEM of 3 to 5 separated experiments * p<0.05; ns, not significant.