Fig. 4: Lipid Raft perturbation by MβCD counteracts FGF2-mediated cell invasion.

PANC-1 and MIA PaCa-2 cells were treated with FGF2 for 24 h at 37 °C in the presence or not of MβCD 2.5 mM. A Western blot analysis shows that the increase of either MCL-1 expression or of SRC phosphorylation (tyr416 site), observed only in PANC-1 cells in response to FGF2, was significantly inhibited by lipid raft perturbation. Densitometric analysis was performed as reported above: *p < 0.05. B Real-time RT-PCR analysis shows that the increase of MMP-2 and MMP-9 mRNA levels, induced by FGF2 stimulation only in PANC-1 cells, is significantly repressed by MβCD treatment. The mRNA levels are expressed as mean value ± SD (n = 3) and reported in the graph as fold increase with respect to the control. Student’s t test was performed, with significance levels defined as p-values < 0.05. p < 0.05: *p < 0.05; ** p < 0.01. C In vitro invasion assay performed in PANC-1 and MIA PaCa-2 cells stably transfected with an unrelated short harpin RNA (PANC-1 Cx shRNA; MIA PaCa-2 Cx shRNA) or with FGFR2c shRNA (PANC-1 FGFR2c shRNA; MIA PaCa-2 FGFR2c shRNA) to obtain FGFR2c depletion. Cells were stimulated with FGF2 in the presence or not of MβCD, as above. The increase of cell invasion in response to FGF2, visible only in PANC-1 cells, was significantly dampened by lipid raft perturbation. The effect of MβCD is comparable to that observed after FGFR2c depletion. No significant effects are visible in MIA PaCa-2 in response to FGF2 stimulation, independently of lipid raft perturbation or FGFR2c depletion. Quantitative analysis was assessed by counting for each sample the migrated cells in 10 microscopic fields from two independent experiments (n = 2). Results were expressed as mean values ± SD. Student’s t test was performed, with significance levels defined as p-values < 0.05. p < 0.05. *p < 0.05. Bar: 20 μm.