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Fig. 3. Src mitogenic signalling is affected by membrane cholesterol depletion and Cav-3DGV expression. (A) CD inhibits PDGF-induced SFK activation. SFKs were immunoprecipitated from RIPA cell lysates, which solubilises caveolae, and subjected to an in vitro kinase assay using enolase as a substrate. In addition to conditions used in Fig. 2, kinase activity was also performed on cells treated with CD and left 2 hours for membrane cholesterol replenishment before PDGF stimulation (Recovery) or not. Top panel: an example of SFK activity (³²P-Enolase); middle panel: quantification of SFK activity under conditions specified (mean ± s.d. from three independent experiments) and expressed as the ratio of non-stimulated and non-treated cells (control); bottom panel: levels of caveolin-1 and tubulin from RIPA and LB cell lysates. Protein levels were assessed by western blotting of total cell lysates using specific antibodies. (B) CD affects Src-specific tyrosine phosphorylation of Stat3. The level of immunoprecipitated Stat3 and pY₇₀₅Stat3 from indicated cell lysates is shown. (C) Membrane cholesterol depletion inhibits PDGF-induced Myc induction. Quiescent cells treated or not with cholesterol-depleting agents in the presence or absence of soluble cholesterol were stimulated for 1 hour with PDGF and total RNA was isolated. Northern blot analysis was performed from indicated RNA using a probe specific for Myc or 26S genes as a control of total RNA level (left panel). Myc mRNA level was quantified by real-time quantitative PCR (right panel). The ratio between the mRNA level and that obtained from quiescent non-treated cells was calculated (Myc response). The mean and the s.d. are shown from three to five independent experiments. (D) SFKs are required for early mitogenic signalling. Quiescent cells were incubated with SU6656 (1 µM) for 1 hour before PDGF stimulation, and subjected to a discontinuous stimulation protocol depicted in top panels. Bottom panels: BrdU incorporation (left) was analysed as described in Fig. 1 and SFK activity (right) was analysed by western blotting of the immunoprecipitated SFK with pY₄₁₆Src antibody specific to the active Src kinases. The ratio between active SFK and SFK levels is shown and is representative of two independent experiments. (E) Expression of Myc overcomes mitogenic inhibition induced by CD. Cells were transfected with a trace amount of GFP construct in the presence of empty vector (mock), and the indicated constructs. Cells were serum starved, treated and stimulated in the presence of BrdU as described in top panel. (F) Expression of Myc overcomes mitogenic inhibition induced by dominant-negative Cav-3DGV. Cells were transfected with trace amounts of GFP construct in the presence of the indicated constructs. Cells were serum starved, and stimulated in the presence of BrdU as described in top panel. 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.

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