Figure 1: Involvement of S1P signalling in PDGF-induced chemotaxis.
(a) SH-SY5Y cells were cultured for 18 hr in the upper chamber with vehicle (control), 10 μM W146, 10 μM JTE-013, 100 ng/ml PTX or 1 μM α-Syn(A53T). The cells migrated into the lower chamber in the absence (none) or presence of 20 ng/ml PDGF were counted. PDGF-induced chemotaxis was expressed as % chemotaxis. Values represent means ± s.e.m. of three independent experiments carried out in triplicate. Statistical significance was analysed by Student’s t-test (**P < 0.01). (b) SH-SY5Y cells transiently transfected with control, S1P1 receptor- or SphK1-siRNA were cultured for 24 hr and then plated on the upper chamber in the absence of serum. The cells migrated into the lower chamber in the absence (none) or presence of 20 ng/ml PDGF were counted. Values represent means ± s.e.m. of 3 independent experiments carried out in triplicate. Statistical significance was analysed by Student’s t-test (**P < 0.01). (c) A schematic diagram for a FRET-based probe to detect conformational changes in S1P1 receptor was depicted. CFP and YFP were separately fused to the same receptor molecule. Under non-activated conditions these two fluoroprobes associate closely (high FRET), whereas S1P-induced conformational changes of the receptor cause their dissociation (low FRET). (d) Cells transiently expressing this FRET probe in (c) were serum-starved for 18 hr and stimulated with 100 nM S1P (first arrow) and analysed for FRET in living cells. Two min after S1P stimulation, 10 μM W146 was added (second arrow). (e) SH-SY5Y cells cotransfected with control or SphK1-siRNA together with vectors encoding this FRET probe in (c) were serum-starved for 18 hr and stimulated with 20 ng/ml PDGF (arrow) and analysed for FRET in living cells. A representative emission ratio of the 2 fluorophores from 5 independent experiments is shown.
