Fig. 4
S1P1 and S1P3 activation mediates the protective effect of ApoM-associated HDL in human endothelium. a-d Cells were pre-incubated with ± W146 1 μM or CAY10444 5 μM in serum/GF starvation medium for 30 min. a HUVEC were incubated in serum/GF starvation medium with total HDL 500 μg/ml and W146 1 μM for 18 h and AnnexinV+ and 7-ADD− cells were quantified by flow cytometry. As control, the experiment was replicated without W146. Error bars indicated SEM of n = 4, Mann-Whitney U-test p = 0.029 when W146 was absent and Mann-Whitney U-test p = 0.343 in W146 presence. b Cells were treated with HDL+ApoM 25 μg/ml plus W146 1 μM for for 2 h and then lysated and Caspase-3 activity measured. Data are represented as the mean ± SEM of n = 2–6. Student t-test p = 0.010 when W146 was not added; Student t-test p = 0.388 in W146 presence. c Cell viability was assayed by MTT. Cells were switched to serum/GF starvation medium with ± HDL+ApoM 25 μg/ml, HDL-ApoM 25 μg/ml and ± W146 1 μM for 24 h. Error bars represent SD of n = 3. Control condition (no W146) data were analyzed by one-way ANOVA p = 0.004 followed by Holm-Sidak method multiple-comparison post hoc test. W146 condition data were analyzed by ANOVA on ranks p = 0.059. d Cells were assayed as in A, but using CAY10444 5 μM instead of W146. Error bars indicated SEM of n = 4-5, Mann-Whitney U-test, p = 0.029 when CAY10444 was not present: Mann-Whitney U-test p = 0.690 in CAY10444 presence. *p < 0.05