Figure 8.

Activation of endogenous S1P receptors by FTY720P induces Mcl-1 to delay the onset of apoptosis in vascular ECs following growth factor withdrawal. (a) HUVECs were switched to serum- and growth factor-free medium in the presence or absence of FTY720P (0.1 μM) for the indicated times before preparation of detergent-soluble cell extracts. Samples were equalised for protein content and fractionated via SDS-PAGE for subsequent immunoblotting with the indicated antibodies. Quantitation of caspase-3 cleavage, Bim expression and Mcl-1 expression at the indicated times normalised to GAPDH is presented as mean values±S.E. for n=3 separate experiments. *P<0.05 versus cells maintained in normal growth medium, ^ΨP<0.05 versus growth factor-deprived cells treated with vehicle. (b) HUVECs were switched to serum- and growth factor-free medium in the presence or absence of FTY720P (0.1 μM) for 3 h before preparation of detergent-soluble cell extracts. Samples were equalised for protein content and fractionated via SDS-PAGE for subsequent immunoblotting with the indicated antibodies. Quantitation of caspase-3 cleavage and Mcl-1 expression normalised to GAPDH is presented as mean values±S.E. for n=3 separate experiments. *P<0.05 versus cells treated without FTY720P at the same time point, ^ΨP<0.05 versus corresponding control siRNA-treated samples