Fig. 2: Lipid raft perturbation by MβCD inhibits FGFR2c oncogenic signaling.

A, B PANC-1 cells were treated with the reported concentrations of methyl-β-cyclodextrin (MβCD) for the different reported times. Cells were then processed for free cholesterol (CHOL) analysis (A) or for MTT assay (B), as reported in materials and methods. A For densitometric analysis of cholesterol levels, the resulting values from two different experiments (n = 2) were expressed as mean values ± SD and reported in graph as fold change respect to the control value. B The cell survival rate was expressed as mean optical density (O.D.) values from three independent experiments (n = 3) ± SD. The concentration of 5 µM for 20 min and 2.5 µM for 24 h were chosen for short-term and long-term experiments, respectively. Student t test was performed and significance levels have been defined as p < 0.05: *p < 0.05; ** p < 0.01; *** p < 0.001. C PANC1 and MIA PaCa-2 cells treated with 5 mM MβCD for 20 min and left untreated or stimulated with FGF2 for 10 min at 37 °C, to induce receptor activation and signaling. Western blot analysis shows the difference in FGFR2 expression, which is not affected by lipid raft perturbation. In addition, only in PDAC-1 cells, lipid raft perturbation represses the phosphorylation of the FGFR2c signaling platform FRS2, that of PKCε and- ERK1/2, as well as that of MTOR and its substrate S6K. MIA PaCa-2 cells, which express very low levels of FGFR2c, display no significant changes in substrate phosphorylation/activation, neither in response to FGF2 or lipid raft perturbation. Densitometric analysis was performed as reported in Fig. 1: *p < 0.05; ** p < 0.01.