Figure 5.

Divergent regulation of Bim accumulation and caspase-3 cleavage in S1P₁-expressing cells following serum withdrawal. (a) Control and S1P₁-expressing CCL39 cells were switched to either serum-free medium (SF) or fresh growth medium (C) for 16 h in the presence or absence of MEK1,2 inhibitor U0126 (10 μM), pertussis toxin (PTX, 100 ng/ml) or vehicle control for 16 h before preparation of cell extracts, fractionation via SDS-PAGE and subsequent immunoblotting with the indicated antibodies. Quantitation of cleaved caspase-3 and Bim expression normalised to GAPDH in control and S1P₁-expressing CCL39 cells is presented as mean values±S.E. for n=3 separate experiments. *P<0.05 versus serum-starved CCL39 control cells, ^ΨP<0.05 versus vehicle-treated CCL39/mycS1P₁ cells. (b) Control and S1P₁-expressing CCL39 cells were switched to SF medium for the indicated times before preparation of detergent-soluble cell extracts. Samples were equalised for protein content before fractionation via SDS-PAGE and subsequent immunoblotting with the indicated antibodies. Quantitation of phospho-ERK1,2 levels normalised to total ERK1,2 is presented as mean values±S.E. for n=3 separate experiments. *P<0.05 versus CCL39 control cells at the indicated time point. (c) Control and S1P₁-expressing CCL39 cells were switched to either SF medium or fresh growth medium (C) for 16 h in the presence of PI3K inhibitor LY294002 (LY, 20 μM) or PKC inhibitor GF109203X (5 μM) either alone or in combination, or a vehicle control for 16 h before preparation of cell extracts, fractionation via SDS-PAGE and subsequent immunoblotting with the indicated antibodies. Quantitation of cleaved caspase-3 and Bim expression normalised to GAPDH in control and S1P₁-expressing CCL39 cells is presented as mean values±S.E. for n=3 separate experiments. *P<0.05 versus serum-starved CCL39 control cells, ^ΨP<0.05 versus vehicle-treated CCL39/mycS1P₁ cells