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Fig. 6. PDGF-induced dorsal ruffle formation and SFK activation outside caveolae require S1P signalling. (A) S1P signalling regulates PDGF-induced dorsal ruffle formation. Left panel: an illustration of a typical effect obtained with S1P signalling inhibition on PDGF-induced dorsal ruffle formation. Right panel: PDGF response (% of cells with dorsal ruffles) of cells treated with indicated drugs or expressing S1K^– as shown. (B) S1P signalling does not affect PDGF-induced BrdU incorporation. Quiescent cells expressing S1K^– or treated with indicated drugs were stimulated with PDGF in the presence of BrdU. BrdU incorporation was analysed and calculated as described in Fig. 1. The mean and the s.d. of three to five independent experiments are shown. (C) S1P signalling regulates PDGF-induced SFK activation in NCF. SFKs were immunoprecipitated from NCF as depicted in Fig. 4 with cells treated or not (control) with indicated agents and stimulated or not with PDGF. Levels of SFKs were determined and the in vitro kinase assay performed with denatured enolase as an exogeneous substrate (³²P-Enolase). (D) S1P signalling does not regulate SFK-PDGFR association in CEF. SFKs were immunoprecipitated from CEF obtained from cells used in panel C and subjected to an in vitro kinase assay. Autophosphorylation of Src kinases (³²P-SFK) as well as the presence of the associated PDGFR (³²P-PDGFR) are shown. Cell treatment was as follows: DMS (10 µM) for 30 minutes, U73343 and U73122 (1 µM) for 30 minutes and PTx (500 ng/ml) for 4 hours.

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